Retinoic acid metabolizing cytochrome p450

ABSTRACT

The present invention provides a novel retinoic acid metabolizing cytochrome P450, P450RAI-3, that is predominantly expressed in the adrenal gland. Methods for and uses of the new polynucleotide, polypeptide, fragments thereof and modulators thereof, include the treatment of cancer.

RELATED APPLICATIONS

This application claim the benefit and priority from U.S. Provisional Patent Application No. 60/292,531 filed May 23, 2001.

FIELD OF THE INVENTION

This invention relates to a novel retinoic acid metabolizing cytochrome P450 and to fragments and variants thereof. It further relates to the nucleic acid encoding said peptides. Products derived or identified using said peptides and nucleic acid molecules of the invention. Methods and uses of said peptides, nucleic acid molecules and products are also encompassed within the scope of said invention.

BACKGROUND OF THE INVENTION

Cytochrome P450s

The cytochromes P450 comprise a large gene superfamily that encodes over 500 distinct heme-thiolate proteins that catalyze the oxidation of drugs and numerous other compounds in the body [Nelson et al., (1996); Guengerich (1991)]. Since there are at least 500 different cytochrome P450 enzymes, it is of considerable interest in the pharmaceutical and other fields to identify which of these enzymes are most important in the metabolism of individual compounds. There are now numerous examples of adverse drug-drug interactions and side effects that can now be understood in terms of the cytochrome P450 enzymes.

P450 proteins are ubiquitous in living organisms, and have been identified in bacteria, yeast, plants and animals [Nelson et al (1996); and Nelson, (1999a)]. The P450 enzymes catalyze the metabolism of a wide variety of drugs, xenobiotics, carcinogens, mutagens, and pesticides, and are responsible for the bioactivation of numerous endogenous compounds including steroids, prostaglandins, bile acids and fatty acids body [Nelson et al., (1996); Guengerich (1991); Nebert et al., (1989)].

Cytochrome P450 metabolism of xenobiotics can result in detoxification of toxic compounds by their conjugation into excretable forms or can result in activation of compounds into metabolites that are toxic, mutagenic, or carcinogenic. Many steroids are deactivated by cytochrome P450-catalyzed oxidation.

Microsomal cytochromes occur on the membrane of the ER and require NADPH cytochrome P450 reductase and a flavoprotein for activity, whereas mitochondrial cytochromes occur on the inner mitochondrial membrane and require ferredoxin and NADPH ferredoxin reductase for activity (Beckman, M., and DeLuca, H. (1997) Methods in Enzymol. 282, 200-223; Armbrecht, H. J., Okuda, K., Wongsurawat, N., Nemani, R., Chen, M., and Boltz, M. (1992) J. Steroid Biochem. Molec. Biol. 43, 1073-1081.) Vitamin A and Retinoic Acid

Vitamin A metabolism gives rise to several active forms of retinoic acid (RA), which are involved in regulating gene expression during development, regeneration, and in the growth and differentiation of epithelial tissues. [Maden, 1992; Chambon, 1995; Mangelsdorf, 1995; Gudas, 1994; Lotan, 1995; Morriss-Kay, 1996] RA has been linked to apoptosis, or programmed cell death in a number of cell types; and to have anticarcinogenic and antitumoral properties [Lotan, 1996].

Early studies of retinol deficiency indicated a correlation between vitamin A depletion and a higher incidence of cancer and increased susceptibility to chemical carcinogenesis [Chytil, 1984]. Several animal models have been used to demonstrate the effectiveness of retinoids in suppressing carcinogenesis in a variety of tissues including skin, mammary epithelia, oral cavity, aerodigestive tract, liver, bladder and prostate [Moon, 1994]. These studies have led to the preventative use of retinoids to treat premalignant lesions including actinic keratosis and oral leukoplakia, as well as in the prevention of secondary tumors of the head and neck and the recurrence of non-small cell lung carcinomas, and basal cell carcinomas [Hong, 1994; Lippman, 1995]. RA itself has been found to be useful therapeutically, notably in the treatment of cancers, including acute promyelocytic leukemia (APL), tumors of the head and neck, and skin cancer, as well as in the treatment of skin disorders such as the premalignancy associated actinic keratoses, acne, psoriasis and ichthyosis. There is evidence that the effectiveness of RA as an anti-tumor agent is at least partially due to induction of cellular differentiation and/or inhibition of proliferation [Lotan, 1996]. Studies over the past several years indicate that a high proportion of patients with acute promyelocytic leukemia (APL) achieve complete remission after a short period of treatment with all-trans RA. Unfortunately, this high rate of remission is in most cases brief. Following relapse, patients are clinically resistant to further treatment with RA [Warrell, 1994; Warrell, et al., 1994; Chomienne, 1996; Muindi, 1992]. The nature of this resistance is unknown. Interestingly, leukemic cells taken from patients exhibiting clinical resistance to RA have been shown to be sensitive to the differentiating action of RA when grown in vitro [Muindi, 1992; Muindi, 1994]. This suggests that pharmacokinetic mechanisms may account for the acquired resistance to RA. This possibility is supported by studies showing that peak plasma concentrations of RA were much higher in patients after initial administration than in patients treated following relapse. This decrease in peak plasma RA concentration was accompanied by a 10-fold increase in urinary 4-oxo-retinoic acid concentration. In addition, ketoconazole, a broad spectrum inhibitor of cytochrome P450 function was shown to modulate RA pharmacokinetics in vivo [Muindi, 1992; Muindi, 1994]. It is therefore likely that RA increases the rate of its own metabolism, which in turn results in the inability to sustain effective therapeutic doses of RA. Therapeutic administration of RA can result in a variety of undesirable side effects and it is therefore important to establish and maintain the minimal requisite doses of RA in treatment. For example, RA treatments during pregnancy can lead to severe teratogenic effects on the fetus. Adverse reactions to RA treatment also include headache, nausea, chelitis, facial dermatitis, conjunctivitis, and dryness of nasal mucosa. Prolonged exposure to RA can cause major elevations in serum triglycerides and can lead to severe abnormalities of liver function, including hepatomegaly, cirrhosis and portal hypertension.

RA metabolism may also account for the lack of response of certain tumors to RA treatment. For example, recent studies have shown that cytochrome P450 inhibitors that block RA metabolism, resulting in increased tissue levels of RA, may be useful therapeutic agents in the treatment of prostate cancer [Wouters, 1992; De Coster, 1996]. Thus RA metabolizing cytochrome P450s may be useful targets for the treatment of a number of different types of cancer.

The classical view of vitamin A metabolism holds that all trans-RA, the most active metabolite is derived from conversion of retinol to retinaldehyde to RA through two oxidation steps and that RA is further metabolized to the polar derivatives 4-OH RA and 4-oxo RA [Blaner, 1994; Napoli, 1995; Formelli, 1996; Napoli, 1996]. It is unknown whether the 4-oxo- and 4-OH— metabolites are simply intermediates in the RA catabolic pathway or whether they can also have specific activities which differ from those of all-trans RA and 9-cis RA. Pijnappel et al. [Pijnappel, 1993] have shown that, in Xenopus, 4-oxo-RA can efficiently modulate positional specification in early embryos and exhibits a more potent ability to regulate Hoxb-9 and Hoxb-4 gene expression than all-trans RA. 4-oxo-RA has been found to bind to retinoic acid receptor-β (RAR-β) with affinity comparable to all-trans RA [Pijnappel, 1993] but poorly to RAR-γ [Reddy, 1992], suggesting that this metabolite exhibits some receptor selectivity. 4-oxo-RA also binds to cellular retinoic acid binding protein (CRABP) but with an affinity slightly lower than that of all-trans RA [Fiorella, 1993]. Takatsuka et al. [Takatsuka, 1996] have shown that growth inhibitory effects of RA correlate with RA metabolic activity but it is unknown whether there is a causal relationship between production of RA metabolites and growth inhibition. The asymmetric distribution of these metabolites in developing embryos suggests that they may be preferentially sequestered or generated by tissue specific isomerases [Creech Kraft, 1994]. The normal balance of these metabolites is dependent upon rate of formation from metabolic precursors, retinol and retinaldehyde [Leo, 1989], and rate of catabolism. Little is presently known about the enzymes involved in this metabolic scheme, in particular the catabolism of RA.

The catabolism of RA is thought to be initiated by hydroxylation either at the C4-, or C18-position of the β-ionone ring of RA [Napoli, 1996]. The C4-hydroxylation step is mediated by cytochrome P450 activity, as judged by the ability of broad spectrum P450 inhibitors such as ketoconazole and liarazole to block 4-hydroxylation [Williams, 1987, Van Wauwe, 1988; Van Wauwe, 1990, Van Wauwe, 1992, Wouters, 1992]. In certain tissues, including testis, skin and lung and in numerous cell lines, such as NIH3T3 fibroblasts, HL 60 myelomonocytic leukemic cells, F9 and P19 murine embryonal carcinoma cell lines and MCF7, RA metabolism can be induced by RA pretreatment [Frolik, 1979, Roberts, 1979a and b; Duell, 1992; Wouters, 1992]. Studies involving targeted disruption of RAR genes in F9 cells suggest that RAR-α and RAR-γ isoforms may play a role in regulating the enzymes responsible for this increased metabolism [Boylan, 1995].

The glucuronidation of RA is a significant metabolic step in the inactivation of RA [Blaner, 1994; Formelli, 1996]. The elimination of RA may require oxidation to 4-oxo, followed by conjugation to form the 4-oxo all-trans RA glucuronide. This is supported by studies in both primates and humans showing that the 4-oxo RA glucuronide is the only retinoid conjugate found in urine [Muindi, 1992; Muindi, 1994]. The fact that following RA therapy, 4-oxo RA is not present or barely detectable in serum, suggests that oxidation may be the rate limiting step in this process.

It has recently been shown that 4-oxoretinol (4-oxo-ROL) can have greater biological activity than retinol. The 4-oxo-ROL is inducible by RA in F9 and P19 mouse teratocarcinoma cells [Blumberg et al., 1995; Achkar et al., 1996].

It is known that zebrafish fins regenerate through an RA sensitive process, which utilizes many gene regulatory pathways involved in early vertebrate development [White, 1994; Akimenko, 1995a & b].

Cytochome P450s and Retinoic Acid Metabolism

In 1979, Roberts et al., [Roberts (1979a)] first postulated that the catabolism of retinoic acid (RA) was mediated by a cytochrome P450 enzyme. Several P450s have since been shown to metabolize RA, including P450 proteins from human, zebrafish and mouse. For example, human P450RAI, which is induced by RA, metabolizes RA to more poplar derivatives including 4-hydroxy retinoic acid (4-OHRA) and 4-oxo retinoic acid (4-oxo RA) [White et al. (1996a)]. Since RA is useful as an antitumor agent, it is desirable to maintain high tissue levels of RA. Thus, cytochrome P450 inhibitors that block RA metabolism, resulting in increased tissue levels of RA, may be useful therapeutic agents in the treatment of cancers, such as prostate cancer [Wouters et al., (1992); and De Coster et al., (1996)].

International Patent Publication No. WO 97/49815, published Dec. 31, 1997, describes a family of retinoid metabolizing proteins, CYP26A, including proteins from human, zebrafish and mouse and their coding sequences. This earlier publication is incorporated herein in its entirety. cDNAs encoding a cytochrome P450-dependent enzyme (P450RAI) which is induced by RA have been cloned and characterized from zebrafish and the protein metabolizes RA to more polar derivatives including 4-hydroxy retinoic acid (4-OH RA) and 4-oxo retinoic acid (4-oxo RA) [White et al., 1996a]. The identification of P450RAI gene is an important step in the understanding of RA signaling but its presence has been known since Roberts et al. (1979a) first postulated that the catabolism of RA was mediated by a P450 enzyme [Frolik et al., 1979; Roberts et al., 1979a]. More recently, the isolation of cDNAs which encode the full-length human and mouse P450RAI orthologs whose expression, like that of the fish cytochrome, is highly inducible by RA has been achieved [Fujii et al., 1997; Ray et al., 1997]. Human and mouse genomic P450RAI-1 sequences and the mouse cDNA sequence encoding P450RAI-1 I have been identified. The human cDNA and amino acid sequence of P450RAI-1 is identified herein as SEQ. ID. NOS. 1 and 2, respectively (also see FIG. 6A). Homologs have also been isolated from human, mouse, chick and xenopus all exhibiting a high degree of sequence conservation [Abu-Abed et al., 1998; Hollermann et al., 1998; White et al., 1997]. There is extensive identity between the human and fish P450RAI genes which overall is 68% at the amino acid level (over 90% between mouse and human).

MCF7 cells have been shown to have RA inducible RA metabolism [Butler and Fontana, 1992; Wouters et al., 1992]. The expression of P450RAI in these cells is dependent on the continuous presence of RA [White et al., 1997]. This suggests that P450RAI regulation by RA forms an autoregulatory feedback loop that functions to limit local concentrations of RA, such that when normal physiological levels of RA are exceeded, induction of P450RAI acts to normalize RA levels. The inducible expression of P450RAI in mouse embryos also suggests that a similar autoregulatory mechanism may limit exposure to RA sensitive tissues during development [lulianella et al., 1999].

A second retinoic acid metabolizing cytochrome P450, P450RAI-2 has also recently been identified in human, rat, mouse and zebrafish. The human cDNA and amino acid sequence are identified herein as SEQ. ID. NOS. 3 and 4, respectively.

Retinoic Acid, Cytochrome p450 and Embryonic Development

All-trans-RA is a critical regulator of gene expression during embryonic development and in the maintenance of adult epithelial tissues [Gudas, et al. (1994).; Lotan, R. M. (1995); Lotan, R. (1996); Morriss-Kay, G. M. (1996)]. The effects of all-trans-RA are mediated by heterodimers of nuclear receptors for retinoic acid (RARs) and retinoid-X-receptors, which are regulated by the 9-cis isomer of RA. Three different subtypes exist for each of these receptors (RARα, β and γ; RXR RAR α, β and γ), which individually are expressed in a tissue specific manner but collectively can be found in essentially all cell types, both during embryonic development and in the adult [Chambon, P. (1995).]. The activity of RA in these tissues is controlled, to a large extent, by enzymes involved in its synthesis from retinaldehyde (ALDH-1 and RALDH-2) and its catabolism to 4-OH, 4-oxo and 18-OH products (P450RAI) [White J. A., et al. (1997); lulianella, A. et al. (1999); McCaffery P. et al., (1999) Niederreither, K. et al. (1999) Swindell E., et al. (1999)].

It has been shown that P450RAI-1 (CYP26A) from zebrafish, mouse, human, chick and xenopus is responsible for the metabolism of active all-trans-RA to inactive polar metabolites including 4-OH-RA, 4-oxo-RA and 18-OH-RA [White J., et al. (1997); Swindell E., et al. (1999); White, J. & Petkovich, M. (1996); Abu-Abed, et al. (1998); Fujii, H. et al. (1997); Ray, W. et al. (1997); Hollermann, T et al. (1998)]. P450RAI-1 expression can be induced by all-trans-RA pre-treatment in multiple tissues, and cell types, and this expression is concomitant with increased all-trans-RA catabolism. In MCF7 cells, all all-trans-RA suggesting a feedback-loop mechanism is dependent on the continued presence of all-trans-RA suggesting a feedback-loop mechanism for the regulation of all-trans-RA levels [White J., et al. (1997)]. Inducible expression of P450RAI-1 has also been observed in vivo in zebrafish, chick, xenopus and mouse embryos suggesting that this autoregulatory feedback-loop plays an important role in balancing all-trans-RA levels in certain developing tissues.

Studies from several groups show that tissues such as neural folds in chick embryos [Swindell E., et al. (1999)], caudal neuroepithelia [lulianella, A et al. (1999); Fujii, H. et al. (1997)] and developing retina [McCaffery P. et al. (1999)] from mouse express P450RAI-1 constitutively thus forming a barrier to all-trans-RA exposure. Comparison of the expression patterns of RALDH-2 and P450RAI-1 in these models suggests that these enzymes act together to form regions of RA synthesis and activity (where RALDH-2 is expressed). RALDH-2 expressing tissues have been shown to contain retinoid activity as measured by both retinoid responsive reporter gene activity and direct measurement of RA levels from tissue extracts; by similar analyses, P450RAI-1 expressing tissues do not [lulianella, A et al. (1999); McCaffery P. et al. (1999)]. In addition, over expression of P450RAI-1 in xenopus embryos has been shown to abrogate the teratogenic effects of exogenously applied RA, consistent with a catabolic role for its enzyme [Hollermann, T et al. (1998)].

The Adrenal Glands

The adrenal glands comprise an inner part (the medulla) that secretes hormones such as adrenaline (epinephrine) that affect blood pressure, heart rate, sweating, and other activities also regulated by the sympathetic nervous system. The outer part (the cortex) secretes many different hormones, including corticosteroids, androgens and minerlocorticoids, which control blood pressure and the levels of salt and potassium in the body.

The adrenal glands are part of a complex system that produces interacting hormones. The hypothalamus produces corticotropin-releasing hormone, triggering the pituitary gland to secrete corticotropin, which regulates the production of orticosteroids by the adrenal glands. Adrenal glands may stop functioning when either the pituitary or hypothalamus fails to produce sufficient amounts of the appropriate hormones. Underproduction or overproduction of any adrenal hormones can lead to serious illness. Diseases associated with the adrenal gland include Addison's disease, Cushing's syndrome, pheochromocytoma, adenoma, hyperaldosteronism, high blood pressure, weakness, paralysis, darkening of the skin, osteoporosis, and fat accumulation.

SUMMARY OF THE INVENTION

The present invention is directed to a novel cytochrome P450 that is part of the retinoic acid metabolizing family of cytochrome P450s. In another aspect, the novel cytochrome P450 is preferentially expressed in the adrenal gland. In another embodiment the novel cytochrome metabolized 13-all-trans-retnoic acid. In yet another embodiment the novel cytochrome metabolized 9-cis-retnoic acid.

The present inventors have characterized for the first time human cytochrome P450RAI-3 [hereinafter “P450RAI-3 or “CYP26C”]. In one embodiment the P450RAI-3 is a microsomal cytochrome. In one embodiment the P450RAI-3 is isolated from adrenal tissue. These findings have important implications in terms of increased understanding of cytochrome P450s and the retinoic acid pathway and the application to various disease states, such as those noted above, i.e. cancer, adenoma, high blood pressure, muscle weakness, skin discolouration, osteoporosis, fat accumulation, pheochromocytoma, Addison's disease, Cushing's syndrome.

Although, the P450RAI-3 and encoding nucleic acid sequence of the invention can be isolated and characterized from any tissue, it is preferably isolated and characterized from adrenal tissue.

Accordingly, the present invention provides an isolated polynucleotide comprising a nucleotide sequence encoding a P450RAI-3, preferably a human P450RAI-3 and to variants, homologs, analogs thereof and to fragments thereof. Complimentary (or antisense) polynucleotide sequences to the polynucleotides of the invention are also encompassed within the scope of the invention.

In a preferred embodiment, an isolated polynucleotide is provided comprising a nucleic acid sequence as shown in SEQ. ID. NOS 9 (FIG. 1) or 10 (FIG. 2). Most preferably, the purified and isolated polynucleotide comprises: (a) a nucleic acid sequence as shown in SEQ. ID. NOS. 9 (FIG. 1) or 10 (FIG. 2) or a nucleic acid sequence encoding the amino acid sequence of SEQ. ID. NO. 3 (FIG. 3), wherein T can also be U; (b) nucleic acid sequences complementary to (a); (c) nucleic acid sequences which are homologous to (a) or (b); or, (d) a fragment of (a) to (c) that will hybridize to (a) to (c) under stringent hybridization conditions. Preferably the fragment is 10 or more, preferably at least 15 bases, most preferably 20 to 30 bases. In another embodiment, the isolated poynucleotide of the invention comprises a sequence encoding any one or more of exons 1 to 6 of P450RAI-3 as depicted in SEQ. ID. NOS 13, 15, 17, 19, 21, or 23 (See FIG. 5 for amino acid regions) or Sequences 12, 14, 16, 18, 20 pr 22 (See FIG. 5). In a further embodiment, the invention provides polynucleotides that consist of the isolated polynucleotides noted herein.

The present invention also includes the P450RAI-3 polypeptide. In one embodiment, the invention provides a polypeptide having an amino acid sequence as shown in SEQ. ID. NO. 11 (FIG. 3) and to variants, homologs, and analogs, insertions, deletions, substitutions and mutations thereto. The invention also comprises polypeptides comprising fragments of the amino acid sequence of SEQ. ID. NO. 11 (FIG. 3) or to their respective variants, homologs, analogs, insertions, deletions, substitutions and mutations. In another embodiment the fragments preferably comprise 14 or more amino acid residues and are most preferably antigenic or immunogenic. In another embodiment the invention provides polypeptides encoded by a polynucleotide having the sequence of SEQ. ID. NO. 10 (FIG. 2), or to variants, homologs, analogs or fragments thereof. In another embodiment the polypeptide of the invention comprises or consists of any one or more of the amino acid sequences of exons 1 to 6 of P450RAI-3 as depicted in SEQ. ID. NOS. 13, 15, 17, 19, 21 or 23 (see FIG. 5).

Accordingly, in one embodiment the invention relates to vectors, host cells comprising the polynucleotides of the invention or that can express the polypeptides of the invention. Antibodies to the polypeptides of the invention are also encompassed within the scope of this invention. The invention further provides recombinant methods for producing P450RAI-3 polypeptides and polynucleotides of the invention. In one embodiment, the invention provides a polynucleotide of the invention operationally linked to an expression control sequence in a suitable expression vector. In another embodiment, the expression vector comprising a polynucleotide of the invention is capable of being activated to express the peptide, which is encoded by the polynucleotide and is capable of being transformed or transfected into a suitable host cell. Such transformed or transfected cells are also encompassed with the scope of this invention.

The invention also provides a method of preparing a polypeptide of the invention utilizing a polynucleotide of the invention. In one embodiment, a method for preparing the polypeptide, preferably P450RAI-3 is provided comprising: transforming a host cell with a recombinant expression vector comprising a polynucleotide of the invention; (b) selecting transformed host cells from untransformed host cells; (c) culturing a selected transformed host cell under conditions which allow expression of the protein; and (d) isolating the protein.

In yet another embodiment, the invention also includes diagnostic methods for detecting and screening for disorders related to P450RAI-3 gene expression and polypeptides and to therapeutic methods for treating such disorders.

As such, the invention also includes a method for detecting a P450RAI-3 related condition in an animal. A P450RAI-3 related condition includes but is not limited to diseases associated with vitamin A or retinoic acid metabolism. The method comprises assaying for P450RAI-3 from a sample, such as a biopsy, or other cellular or tissue sample, from an animal susceptible of having such a condition. In one embodiment, the method comprises contacting the sample with an antibody of the invention, which binds P450RAI-3, and measuring the amount of antibody bound to P450RAI-3 in the sample, or unreacted antibody. In another embodiment, the method involves detecting the presence of a nucleic acid molecule having a sequence encoding a P450RAI-3, comprising contacting the sample with a nucleotide probe which hybridizes with the nucleic acid molecule, preferably mRNA or cDNA to form a hybridization product under conditions which permit the formation of the hybridization product, and assaying for the hybridization product.

The invention further includes a kit for detecting a P450RAI-3 related condition from a sample comprising an antibody of the invention, preferably a monoclonal antibody. Preferably directions for its use are also provided. The kit may also contain reagents, which are required for binding of the antibody to a P450RAI-3 protein in the sample.

The invention also provides a kit for detecting the presence of a polypeptide having a sequence encoding a polypeptide of, related to or analogous to a polypeptide of the invention, comprising a nucleotide probe which hybridizes with the nucleic acid molecule, reagents required for hybridization of the nucleotide probe with the nucleic acid molecule, and directions for its use.

The invention also includes screening methods for identifying binding partners of P450RAI-3. In addition, the invention relates to screening methods for identifying modulators, such as agonists and antagonists, of P450RAI-3 activity. In one embodiment such modulators of P450RAI-3 activity or expression can include antibodies to P450RAI-3 and antisense polynucleotides to the P450RAI-3 gene or fragment thereof.

The invention further provides a method of treating or preventing a disease associated with P450RAI-3 expression comprising administering an effective amount of an agent that activates, simulates or inhibits P450RAI-3 expression, as the situation requires, to an animal in need thereof. In a preferred embodiment, P450RAI-3, a therapeutically active fragment thereof, or an agent, which activates or simulates P450RAI-3 expression is administered to the animal in need thereof to treat a disease or condition associated with too much retinoic acid. In another embodiment the disease is associated with over expression of P450RAI-3 or retinoic acid deficiency (i.e. not enough retinoic acid or desire to maintain retinoic acid levels) and the method of treatment comprises administration of an effective amount of an agent that inhibits P450RAI-3 expression such as an antagonist of P450RAI-3, an antibody to P450RAI-3, a mutation thereof, or an antisense nucleic acid molecule to all or part of the P450RAI-3 gene.

In another embodiment the invention provides pharmaceutical compositions comprising a modulator of P450RAI-3 activity and a pharmaceutical acceptable carrier. In another embodiment, the pharmaceutical composition of the invention comprises P450RAI-3 (preferably a soluble form thereof) or a therapeutically effective fragment thereof and a pharmaceutically acceptable carrier. In another embodiment the pharmaceutical compositions of the invention comprise both a modulator of P450RAI-3 activity and P450RAI-3 (preferably a soluble form thereof) or a therapeutically effective fragment thereof. In a further embodiment the pharmaceutical compositions of the invention can further comprise a modulator of P450RAI-3 and any one or more of: (a) retinoic acid, (b) a ligand of P450RAI-3; a substrate of P450RAI-3.

The invention also includes a method of identifying a modulator of P450RAI-3 activity comprising:

-   -   [a]incubating P450RAI-3 or a cell expressing P450RAI-3 with a         test compound under conditions that promote P450RAI-3 expression         or activity;     -   [b] detecting the activity or expression, as the case may be, of         P450RAI-3 in the presence of said test compound, a decrease in         said activity or expression being indicative that the test         compound is an inhibitor of P450RAI-3 expresssion or activity,         while an increase in said expression or activity is indicative         that the test compound is a P450RAI-3 agonist.

Another aspect of the invention relates to a method of identifying a substate of P450RAI-3 comprising:

-   -   [a]incubating P450RAI-3 with a test substrate under conditions         that promote P450RAI-3/substate complex formation or         interaction;     -   [b] determining P450RAI-3/substrate complex formation or         interaction.         The incubation step optionally further comprises a known         modulator of P450RAI-3. Step [b] can be determined by comparing         the effect on P450RAI-3 in the absence and presence of the test         substrate.

Another aspect of the invention is a method for identifying a substance which associates with a protein of the invention comprising

-   -   (a) reacting the protein with at least one substance which         potentially can associate with the protein, under conditions         which permit the association between the substance and protein,         and     -   (b) removing or detecting protein associated with the substance,         wherein detection of associated protein and substance indicates         the substance associates with the protein.

Another embodiment of the invention relates to a method for evaluating a compound for its ability to modulate the biological activity of a protein of the invenition comprising providing the protein with a substance which associates with the protein and a test compound under conditions which permit the formation of complexes between the substance and protein, and removing and/or detecting complexes.

The invention also relates to a method for identifying inhibitors of a P450RAI-3 Protein interaction, comprising

-   -   (a) providing a reaction mixture including the P450RAI-3 Related         Protein and a substance that binds to the P450RAI-3 Related         Protein, or at least a portion of each which interact;     -   (b) contacting the reaction mixture with one or more test         compounds;     -   (c) identifying compounds which inhibit the interaction of the         P450RAI-3 Related Protein and substance.

The invention also includes a method for detecting a nucleic acid molecule encoding a protein comprising an amino acid sequence of SEQ. ID. NO. 40 in a biological sample comprising the steps of:

-   -   (a) hybridizing a nucleic acid molecule of claim 1 to nucleic         acids of the biological sample, thereby forming a hybridization         complex; and     -   (b) detecting the hybridization complex wherein the presence of         the hybridization complex correlates with the presence of a         nucleic acid molecule encoding the protein in the biological         sample.

The invention also includes a method as claimed in claim 50 wherein nucleic acids of the biological sample are amplified by the polymerase chain reaction prior to the hybridizing step.

(a) The invention also includes a composition comprising one or more of a nucleic acid molecule or protein claimed in any of the preceding claims or a substance or compound identified using a method, and a pharmaceutically acceptable carrier, excipient or diluent. The invention also includes a nucleic acid molecule or protein of the invention, or a substance or compound identified using a method of the invention in the preparation of a pharmaceutical composition for treating a condition mediated by a protein of the invention, or a nucleic acid molecule of the invention.

The invention includes compounds identified with methods of the invention.

Another aspect of the invention includes a vaccine for stimulating or enhancing in a subject to whom the vaccine is administered production of antibodies directed against a protein of the invention. The invention also includes a method for stimulating or enhancing in a subject production of antibodies directed against a protein. The invention includes a method involving administering to the subject a vaccine of the invention in a dose effective for stimulating or enhancing production of the antibodies.

The invention includes the use of the isolated polypeptide of the invention, and optionally a modulator of P450RAI-3 activity for preparation of a pharmaceutical substance. The invention also includes the use of a therapeutically effective amount of the polypeptide of the invention or of the polynucleotide of the invention and/or a modulator of P450RAI-3 for preventing, treating or ameliorating a medical condition related to P450RAI-3 expression.

The invention includes the use of a therapeutically effective polypeptide of the invention and/or an agonist thereof for treating a disease or condition related to vitamin A or retinoic acid metabolism in a patient. The invention also includes the use of a P450RAI-3 inhibitor and a P450RAI-3 substrate for preventing, treating or ameliorating a medical condition related to P450RAI-3 expression or for preparation of a pharmaceutical substance.

Another embodiment of the invention relates to a method of determining the ATRA and/or 9-cis-RA metabolizing activity of a polypeptide of the invention, comprising: expressing the polypeptide in a host cell, adding ATRA and/or 9-cis-RA to the cell, and determining the amount and/or rate of ATRA and/or 9-cis-RA metabolism.

Another embodiment of the invention relates to a method of determining the substrate of a polypeptide of the invention, comprising: expressing the polypeptide in a host cell, adding a candidate substrate, and determining if the substrate is metabolized, wherein metabolization indicates that the candidate substrate is a substrate of the polypeptide.

The invention also relates to a method of determining the binding activity of a substrate to a polypeptide of the invention, comprising expressing the polypeptide in a host cell, adding a candidate substrate, and determining a Kd value.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only. Various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the 22,179 bp genomic DNA sequence of P450RAI-3 [SEQ. ID. NO. 9] based on contig information from AL358613 and the sequence obtained from the Centre for Applied Genomics. The corresponding amino acid sequence (from three different reading frames of the nucleotide sequence) are also shown.

FIG. 2 is the 1569 bp cDNA sequence of P450RAI-3 [SEQ. ID. NO. 10].

FIG. 3 is the 522 amino acid sequence of P450RAI-3 [SEQ. ID. NO. 11].

FIG. 4 is the nucleotide and amino acid sequence alignment of P450RAI-3.

FIG. 5 is a schematic diagram illustrating the 6 exons of human cytochrome P450RAI-3 on human genomic clone (SEQ. ID. NO. 9). The numbers above the schematic diagram indicate the amino acid regions of the exons with respect to FIG. 3 [SEQ. ID. NO. 11] and the numbers below the schematic refer to the nucleotide positions on the human genomic clone sequence (SEQ. ID. NO. 9, or FIG. 1).

FIG. 6A shows the amino acid sequence alignment of P450RAI-1, P450RAI-2 and P450RAI-3. FIG. 6B shows the alignment score amongst the three cytochrome P450RAIs on an amino acid level. FIG. 6C shows the alignment score amongst the three cytochrome P450RAIs at a nucleic acid level.

FIG. 7A is a dot-blot illustrating expression of P450RAI-3 in various human tissues. FIG. 7B is the legend of the dot blot of FIG. 6A.

FIG. 8 is a 1% agarose gel illustrating expression of P450RAI-3 in various human tissues.

FIG. 9 is a Southern blot illustrating expression of P450RAI-3 in the human adrenal gland.

FIGS. 10A and B are bar graphs illustrating all trans retinoic acid activity (radioactivity counts) for aqueous (A) and organic (B) fraction of mammalian Cos-1 cells transfected with either pcDNA-P450RAI-3 or pcDNA-control vector. Results for Cos cells alone and NCC are also provided.

FIG. 11 illustrates the HPLC analysis of organic metabolites from ATRA metabolism assay showing that P450RAI-3 (Cos-pcDNA-CYP26C) (B) metabolizes ATRA to polar metabolites, whereas no such metabolic activity is seen in the control (Cos-pcDNA) (A).

FIGS. 12A and B are linear graphs, illustrating the results of the substrate assay of Example 4. Determination of P450RAI-3 substrates among the candidate compounds ATRA, RA-precursors (Retinol, Retinal), RA-isomers (13-cis and 9-cis forms), or RA-metabolites (40H-RA, 4-OXORA, and 18-OHRA).

FIGS. 13A and B are linear graphs illustrating the comparison of [³H]-RA-metabolism inhibition with increased concentration of unlabeled ATRA, 9-cis-RA, 13-cis-RA and Ketoconazole in transient Cos-P450RAI-3 (A) vs. Cos-CYP26-B (B) cell-based assay.

FIG. 14A is a bar graph illustrating an assessment of RA-metabolism in P450RAI-3 (CYP26C) and CYP26-B-infected Sf9-cells. FIG. 14B is illustrated P450RAI-3 activity in the presence and absence of NADPH. The results are consistent with P450RAI-3 being a microsomal cytochrome P450.

FIGS. 15A and B are linear graphs illustrating the comparison of [³H]-RA-metabolism inhibition with increased concentration of either unlabeled ATRA, 9-cis-RA, 13-cis-RA or Ketoconazole in Sf9-Bac-P450RAI-3 (A) and in Sf9-Bac-CYP26-B (B) insect microsomes.

FIGS. 16A and 16B are linear graphs illustrating the results of the [³H]-RA-metabolism inhibition assay carried out with increased concentration of unlabeled ATRA, 13-ci-sRA and 9-cis-RA isomers in P450RAI-3 (A) and CYP26-B (B) mammalian microsomes.

FIGS. 17A (linear graph) and B (Lineweaver-Burk plot) are graphs illustrating results of ATRA/P450RAI-3 and CYP26B binding assay of Example 7 using Sf9-Bac-P450RAI-3 and Sf9-Bac-CYP26B insect microsomes.

FIGS. 18A (linear graph) and B (Lineweaver-Burk plot) are graphs illustrating results of 9-cis RA/CYP26C (P450RAI-3) binding assay of Example 7. Determination of binding of 9-cis-RA to CYP26-A, B, and C was carried out at substrate concentrations of 0.05 nM to 1000 nM but it was not possible to determine the kD values for CYP26A and CYP26B (See Example 7).

FIGS. 19A and B are bar graphs illustrating the analysis of water soluble (A) and organic(B)metabolites from 9-cis-RA-metabolism in P450RAI-3 transient transfected Cos cells as-per Example 8.

FIGS. 20A and B illustrate the HPLC analysis of 9-cis-RA metabolism in control (A) and P450RAI-3 (B) transient transfected Cos cells.

FIGS. 21A and B illustrate the mass spectrometry analysis of ATRA (B) and 9-cis-RA (A) metabolism by Hela-P450RAI-3 stable cells as per Example 9.

FIGS. 22A and B are graphs illustrating the assessment of ATRA (A) and 9-cis-RA(B) metabolism in Hela cell clones (pcEBVclone #1, pcDNA-clone #22).

FIG. 23 is a graph illustrating RA metabolic activity in microsomes made from Hela-P450RAI-3 clone 1 as per Example 10.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See for example, Sam brook, Fritch, & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); DNA Cloning: A practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization B. D. Hames & S. J. Higgens eds. (1985); Transcription and Translation B. D. Hames & S. J. Higgins eds (1984); Animal Cell Culture R. I. Freshney, ed. (1986); Immobilized Cells and enzymes IRL Press (1986); and B. Perbal, A practical Guide to Molecular Cloning (1984).

The following definitions are provided to facilitate understanding of certain terms used in this application.

Abbreviations for amino acid residues are the standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 common L-amino acids. Likewise abbreviations for nucleic acids are the standard codes used in the art.

In the present invention, “isolated” refers to material removed from its original environment [e.g., the natural environment if it is naturally occurring], and thus is altered “by the hand of man” from its natural state. For example, an isolated polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be “isolated” because that vector, composition of matter, or particular cell is not the original environment of the polynucleotide. However, isolated polynucleotides do not include chromosomes in the present invention.

In the present invention a “secreted” protein refers to a protein capable of being directed to the ER, secretory vesicles, or the extracellular space as a result of a signal sequence, as well as a protein released into the extracellular space without necessarily containing a signal sequence. If the secreted protein is released into the extracellular space, the secreted protein can undergo extracellular processing to produce a mature protein. Release into the extracellular space can occur by many mechanisms, including exocytosis and proteolytic cleavage.

The term “agonist” of a polypeptide of interest, refers to a compound that interacts with the polypeptide e.g. P450RAI-3, either directly or indirectly and maintains or increases the activity of the polypeptide. Agonists may include proteins, peptides, nucleic acids, carbohydrates, or any other molecules. Agonists also include a molecule derived from P450RAI-3 or a substrate or ligand thereto. Peptide mimetics, synthetic molecules with physical structures designed to mimic structural features of particular peptides, may serve as agonists. The stimulation may be direct, or indirect, or by a competitive or non-competitive mechanism.

The term “antagonist”, as used herein, of a polypeptide of interest, for example P450RAI-3, refers to a compound that does not maintain or inhibits the activity of the polypeptide. Antagonists may include proteins, peptides, nucleic acids, carbohydrates, or any other molecules. Antagonists also include a molecule derived from a P450RAI-3 or a substrate or ligand thereto. Peptide mimetics, synthetic molecules with physical structures designed to mimic structural features of particular peptides, may serve as antagonists. The inhibition may be direct, or indirect, or by a competitive or non-competitive mechanism.

“Peptide mimetics” or “peptidomimetics” are structures, which serve as substitutes for peptides in interactions between molecules (See Morgan et al (1989), Ann. Reports Med. Chem. 24:243-252 for a review). Peptide mimetics include synthetic structures, which may or may not contain amino acids and/or peptide bonds but retain the structural and functional features of a peptide, or agonist or antagonist (i.e. enhancer or inhibitor) of the invention. Peptide mimetics also include peptoids, oligopeptoids (Simon et al (1972) Proc. Natl. Acad, Sci USA 89:9367); and peptide libraries containing peptides of a designed length representing all possible sequences of amino acids corresponding to a motif, peptide, or agonist or antagonist (i.e. enhancer or inhibitor) of the invention.

The terms “interact”, “interaction”, or “interacting” refer to any physical association between proteins, other molecules such as lipids, carbohydrates, nucleotides, and other cell metabolites. Examples of interactions include protein-protein interactions, protein-lipid interactions, and lipid-lipid interactions. The term preferably refers to a stable association between two molecules due to, for example, electrostatic, hydrophobic, ionic and/or hydrogen-bond interactions under physiological conditions. Certain interacting or associated molecules interact only after one or more of them has been stimulated (e.g. phosphorylated). An interaction between proteins and other cellular molecules may be either direct or indirect. Various methods known in the art can be used to measure the level of an interaction. For example, the strength of covalent bonds may be measured in terms of the energy required to break a certain number of bonds.

“P450RAI-3” or “CYP26C” as used herein both refer to the novel cytochrome P450 retinoic acid inducible polypeptide of the invention.

ii) P450RAI-3 Polynucleotides and Polypeptides

The present invention provides a novel cytochrome P450 polypeptide, P450RAI-3, and polynucleotide encoding the same. Fragments and modifications (or variants) to the polypeptide and polynucleotide of the novel cytochrome and obvious chemical equivalents thereof, are also encompassed within the scope of the present invention.

The present inventors isolated human P450RAI-3 and the encoding polynucleotide. P450RAI-3 showed expression in the adrenal gland. The genomic nucleotide sequence or P450RAI-3 is shown in FIG. 1 [SEQ. ID. NO. 9]. The cDNA coding sequence is shown in FIG. 2 [SEQ. ID. NO. 10]. It is 1569 nucleotides in length and contains an open reading frame encoding a polypeptide of 522 amino acid residues, the sequence of which is shown in FIG. 3[SEQ. ID. NO. 11]. Certain exons [exons 1, 4, 5, and 6] of P450RAI-3 were first identified on genomic clone AL358613.4, a 160,532 bp long polynucleotide sequence. The present inventors have identified the whole cDNA sequence and determined its function.

Elevated P450RAI-3 protein expression was observed in the human adrenal gland. These results suggest P450RAI-3 may have a role in retinoic acid metabolism in the adrenal gland and associated conditions.

P450RAI-3 has application to general physiological processes including various conditions such as those related to retinoic acid metabolism. P450RAI-3 metabolizes all-trans-retinoic-acid (ATRA) to polar metabolites. 9-cis-RA competes strongly for P450RAI-3 activity (ID₅₀1 μM) while ketoconazole is a weak inhibitor (ID₅₀ 70 μM). P450RAI-3 metabolies ATRA as well as 9-cis-RA to hydroxy and oxo-metabolites. Retinol (Vitamin A) and Retinal are not substrates for CYP26-C.

P450RAI-3 has been shown to be about 43% identical at the amino acid level with P450RAI-1 and 51% identical to P450RAI-2. At the nucleic acid level, P450RAI-3 is 52% and 61% homologous to P450RAI-1 and P450RAI-2, respectively.

The cytochrome P450s are heme-binding proteins that contain the putative family signature F(XX)G(XXX)C(X)G (X means any residue; conserved residues are in bold (SEQ. ID. NO. 1)). (Nelson, D. R., Methods in Molecular Biology, Vol. 107: Cytochrome P450 Protocols, Cytochrome P450 Nomenclature, pp. 15-24, Phillips, I. R. and Shephard, E. A., eds., Humana Press Inc., Totowa, N. J. (1998)). The heme-binding signature in P450RAI-3 can be found at amino acids 452-461 of FIG. 3 and contains the motif FGGGARSCLG. (SEQ. ID. NO. 8)

Heme-binding proteins, such as myoglobin, hemoglobin and cytochromes, play an important role in several cellular processes, such as respiration and detoxification. For example, the capacity of myoglobin or hemoglobin to bind oxygen depends on the presence of a heme group. Heme consists of an organic part and an iron atom. The iron atom in heme alternates between a ferrous (+2) and a ferric(+3) state; however, only heme containing an iron atom in the +2 oxidation state binds oxygen. (For a review, see e.g., Stryer, Biochemistry (3^(rd) edition) W.H. Freeman and Co., New York, pp. 144 and 404-405 (1988).)

Cytochrome P450s play an important role in the detoxification of toxic substances (xenobiotics), such as phenobarbital, codeine and morphine, by oxidation. It is the ability of P450s to bind heme and oxygen that enables them to function as oxidative enzymes. (for a review, see e.g. Darnelle et al., Molecular Cell Biology (2^(nd) edition), W.H. Freemand and Co., New York, pp 397 and 981-982 (1990)). Thus peptides of P450RAI-3 containing the heme-binding motif or the oxygen binding domain and related activities and functions are also contemplated by the invention.

The P450RAI-3 cDNA sequence (SEQ. ID. NO. 10), such as in FIG. 2, is useful for designing nucleic acid hybridization probes that will detect nucleic acid sequences contained in P450RAI-3 cDNA sequence. These probes will also hybridize to nucleic acid molecules in biological samples, thereby enabling a variety of forensic and diagnostic methods of the invention. Similarly, polypeptides identified from the P450RAI-3 amino acid sequence, such as disclosed in FIG. 3 (SEQ. ID. NO. 11), may be used to generate antibodies, which bind specifically to P450RAI-3.

Nonetheless, DNA sequences generated by sequencing reactions can contain sequencing errors. The errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence. The erroneously inserted or deleted nucleotides cause frame shifts in the reading frames of the predicted amino acid sequences. In this case, the predicted amino acid sequences diverge from the actual amino acid sequences, even though the generated DNA sequences may be greater than 99.9% identical to the actual DNA sequence. For example, one base insertion or deletion in an open reading frame of over 1000 bases.

For those applications requiring precision in the nucleotide sequence or the amino acid sequence, the present invention provides the generated nucleotide sequence of P450RAI-3 as depicted in FIG. 2 (SEQ. ID. NO. 10) and the predicted translated amino acid sequence of FIG. 3 ((SEQ. ID. NO. 11).

The present invention also relates to the P450RAI-3 gene and the gene corresponding to FIG. 1 (genomic DNA (SEQ. ID. NO. 9)) or 2 (cDNA (SEQ. ID. NO. 10)). The P450RAI-3 gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include preparing probes or primers from the disclosed sequence and identifying or amplifying the P450RAI-3 gene from appropriate sources of genomic materials.

Also provided in the present invention are species homologs of human P450RAI-3. Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for the desired homolog.

As used herein and encompassed within the scope of this invention, a P450RAI-3 “nucleic acid” or “nucleic acid molecule” or “polynucleotide” refers to a molecule having the nucleic acid sequence as shown in FIG. 1 (SEQ. ID. NO. 9)or the coding region thereof, such as the sequence of FIG. 2 (SEQ. ID. NO. 10). For example, P450RAI-3 polynucleotide can contain the nucleotide sequence of the full length cDNA sequence as well as fragments, epitomes, domains and variants of the nucleic acid. Furthermore, a P450RAI-3 “polypeptide” as used herein refers to a molecule having the translated amino acid sequence generated from the polynucleotide as broadly defined and preferably having the sequence of FIG. 3 (SEQ. ID. NO. 11).

A P450RAI-3 “polynucleotide” also refers to isolated polynucleotides, which encode the P450RAI-3 polypeptides and to polypeptides closely related thereto.

A P450RAI-3 “polynucleotide” also refers to isolated polynucleotides, which encode the amino acid sequence in FIG. 3 (SEQ. ID. NO. 11), or a biochemically active fragment thereof, including obvious chemical equivalents thereof.

A P450RAI-3 polynucleotide also encompasses those polynucleotides which differ from any of the polynucleotides of the invention in codon sequence due to the degeneracy of the genetic code such polynucleotides encode functionally equivalent polypeptides but differ in sequence from the above mentioned sequences due to degeneracy in the genetic code.

A P450RAI-3 “polynucleotide” also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to polynucleotide sequences disclosed herein, such as those of FIG. 1 (SEQ. ID. NO. 9) or 2 (SEQ. ID. NO. 10) or the complement thereof. “Stringent hybridization conditions” refers to an overnight incubation at 42° C. in a solution comprising 50% formamide, 5×SSC [750 mM NaCl, 75 mm sodium citrate], 50 mM sodium phosphate [pH 7.6], 5× Denhardt's solution, 10% dextran sulfate, and 20 ug/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC at about 65° C.

Of course, a polynucleotide which hybridizes only to poly A+ sequences [such as any 3′ terminal poly A+ tract of a cDNA] or to a complementary stretch of T [or U] residues, would not be included in the definition of “polynucleotide”, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly [A] stretch or the complement thereof [e.g., practically any double-stranded cDNA clone].

The P450RAI-3 polynucleotide can be composed of any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. As such, in the sequences referred to herein “T” can also be “U”. For example, P450RAI-3 polynucleotide can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double stranded regions. In addition, the P450RAI-3 polynucleotide or hybrid thereof can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA.

In addition, P450RAI-3 polynucleotide may contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. “Modified” bases include, for example, tritiated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically, or metabolically modified forms. Modified forms also encompass analogs of the polynucleotide sequence of the invention, wherein the modification does not alter the utility of the sequences described herein. In one embodiment, the modified sequence or analog may have improved properties over unmodified sequence.

One example of a modification to prepare an analog within the scope of this invention is to replace one of the naturally occurring bases (i.e. adenine, guanine, cytosine or thymidine) of the sequence shown in FIG. 1 or 2 (SEQ. ID. NOS. 9 or 10) with a modified base such as such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8 amino guanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxyl guanine and other 8-substituted guanines, other aza and deaza uracils, thymidines, cytosines, adenines, or guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosine.

Another example of a modification is to include modified phosphorous or oxygen heteroatoms in the phosphate backbone, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages in the nucleic acid molecule shown in FIG. 1 or 2 (SEQ. ID. NOS. 9 or 10). For example, the nucleic acid sequences may contain phosphorothioates, phosphotriesters, methyl phosphonates, and phosphorodithioates.

A further example of an analog of a nucleic acid molecule of the invention is a peptide nucleic acid (PNA) wherein the deoxyribose (or ribose) phosphate backbone in the DNA (or RNA), is replaced with a polyamide backbone which is similar to that found in peptides (P. E. Nielsen, et al Science 1991, 254, 1497). PNA analogs have been shown to be resistant to degradation by enzymes and to have extended lives in vivo and in vitro. PNAs also bind stronger to a complimentary DNA sequence due to the lack of charge repulsion between the PNA strand and the DNA strand. Other nucleic acid analogs may contain nucleotides containing polymer backbones, cyclic backbones, or acyclic backbones. For example, the nucleotides may have morpholino backbone structures (U.S. Pat. No. 5,034,506). The analogs may also contain groups such as reporter groups, a group for improving the pharmacokinetic or pharmacodynamic properties of nucleic acid sequence.

The P450RAI-3 polypeptide of the invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. The P450RAI-3 polypeptides may be modified by either natural processes, such as post-translational processing or by chemical modification techniques, which are well known in the art. Such modifications are described in basic texts, research manuals and research literature. Modifications may occur anywhere in the P450RAI-3 including the peptide backbone, the amino acid side-chain and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degree at several sites in a given P450RAI-3 polypeptide. In addition, a given P450RAI-3 may contain many types of modification. The modifications may result from post-translational natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulphide bond formation, demethylation, formation of covalent cross-links, reduction of disulphide bonds into free cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulphation, transfer-RNA mediated addition of amino acids to proteins such as arginylation and ubiquitination [See, for example, Proteins-Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W.H. Freeman and Company, New York (1993); Post-Translational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, p 1-12 (1983); Seifter et al., Methods in Enzymology 182: 626-646 (1990); Rattan et al., Ann. NY Acad. Sci. 663: 48-62 (1992).]

A P450RAI-3 exhibiting activity similar, but not necessarily identical to, an activity of a P450RAI-3 polypeptide, including mature forms, as measured by a given biological assay, with or without dose dependency are also encompassed within the scope of this invention. Where dose dependency exists, it need not be identical to the P450RAI-3 polypeptide but rather substantially similar to the dose-dependency in a given activity as compared to the P450RAI-3 polypeptide. For example, the candidate polypeptide will exhibit greater activity or not more than about 25-fold less and, preferably, not more than about tenfold less activity and most preferably, not more than about three-fold less activity relative to P450RAI-3 polypeptide.

P450RAI-3 polypeptide thereof may include various structural forms of the primary protein that retain biological activity. For example, a polypeptide of the invention may be in the form of acidic or basic salts or in neutral form. The polypeptides of the invention may be in the form of a secreted protein (i.e. could include fusion proteins or solubulized forms of the proteins of the invention), including the mature form or may be part of a larger protein such as a fusion protein. It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification, such as multiple histidine residues, or an additional sequence for stability during recombinant production.

P450RAI-3 polypeptides are preferably provided in an isolated form, and preferably are substantially purified, A recombinantly produced version of a P450RAI-3 polypeptide (including the secreted polypeptide, if genetically modified to be a secreting protein), can be substantially purified by the one-step method described in Smith and Johnson, Gene 67: 31-40 (1988). P450RAI-3 polypeptides also can be purified from natural or recombinant sources using antibodies of the invention raised against the polypeptides of the invention in methods well known in the art.

Production of Polynucleotides and Polypeptides of the Invention

The polynucleotides and polypeptides of the invention can be prepared in any suitable manner, such means being known to persons skilled in the art. Such methods include isolating naturally occurring polypeptides and polynucleotides, recombinantly or synthetically/chemically produced polynucleotides or polypeptides or a combination of these methods.

An isolated nucleic acid molecule of the invention which comprises DNA can be isolated by preparing a labelled nucleic acid probe based on all or part of the nucleic acid sequences as shown in FIG. 1 or 2 (SEQ. ID. NOS. 9 or 10) and using this labelled nucleic acid probe to screen an appropriate DNA library (e.g. a cDNA or genomic DNA library). For example, a genomic library isolated can be used to isolate a DNA encoding a novel protein of the invention by screening the library with the labelled probe using standard techniques. Nucleic acids isolated by screening of a cDNA or genomic DNA library can be sequenced by standard techniques.

An isolated nucleic acid molecule of the invention, which is DNA, can also be isolated by selectively amplifying a nucleic acid encoding a novel protein of the invention using the polymerase chain reaction (PCR) methods and cDNA or genomic DNA. It is possible to design synthetic oligonucleotide primers from the nucleic acid sequence as shown in FIG. 1 or 2 (SEQ. ID. NOS. 9 or 10) for use in PCR. A nucleic acid can be amplified from cDNA or genomic DNA using these oligonucleotide primers and standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. It will be appreciated that cDNA may be prepared from mRNA, by isolating total cellular mRNA by a variety of techniques, for example, by using the guanidinium-thiocyanate extraction procedure of Chirgwin et al., Biochemistry, 18, 5294 5299 (1979). cDNA is then synthesized from the mRNA using reverse transcriptase (for example, Moloney MLV reverse transcriptase available from Gibco/BRL, Bethesda, Md., or AMV reverse transcriptase available from Seikagaku America, Inc., St. Petersburg, Fla.).

An isolated nucleic acid molecule of the invention, which is RNA, can be isolated by cloning a cDNA encoding a novel protein of the invention into an appropriate vector, which allows for transcription of the cDNA to produce an RNA molecule, which encodes a protein of the invention. For example, a cDNA can be cloned downstream of a bacteriophage promoter, (e.g., a T7 promoter) in a vector, cDNA can be transcribed in vitro with T7 polymerase, and the resultant RNA can be isolated by standard techniques.

A nucleic acid molecule of the invention may also be chemically synthesized using standard techniques. Various methods of chemically synthesizing polydeoxynucleotides are known, including solid-phase synthesis, which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers (See e.g., Itakura et al. U.S. Pat. No. 4,598,049; Caruthers et al. U.S. Pat. No. 4,458,066; and Itakura U.S. Pat. Nos. 4,401,796 and 4,373,071).

Determination of whether a particular nucleic acid molecule encodes a novel protein of the invention may be accomplished by expressing the cDNA in an appropriate host cell by standard techniques, and testing the activity of the protein using the methods as described herein. A cDNA having the activity of a novel protein of the invention so isolated can be sequenced by standard techniques, such as dideoxynucleotide chain termination or Maxam-Gilbert chemical sequencing or by automated DNA sequencing, to determine the nucleic acid sequence and the predicted amino acid sequence of the encoded protein.

The initiation codon and untranslated sequences of nucleic acid molecules of the invention may be determined using currently available computer software designed for the purpose, such as PC/Gene (IntelliGenetics Inc., Calif.). Regulatory elements can be identified using conventional techniques. The function of the elements can be confirmed by using these elements to express a reporter gene, which is operatively linked to the elements. These constructs may be introduced into cultured cells using standard procedures. In addition to identifying regulatory elements in DNA, such constructs may also be used to identify proteins interacting with the elements, using techniques known in the art.

The sequence of a nucleic acid molecule of the invention may be inverted relative to its normal presentation for transcription to produce an antisense nucleic acid molecule. The term “antisense” nucleic acid molecule is a nucleotide sequence that is complementary to its target. Preferably, an antisense sequence is constructed by inverting a region preceding or targeting the initiation codon or an unconserved region. In another embodiment the antisense sequence targets all or part of the mRNA or cDNA encoding P450RAI-3. In particular, the nucleic acid sequences contained in the nucleic acid molecules of the invention or a fragment thereof, preferably a nucleic acid sequence shown in FIG. 1 or 2 (SEQ. ID. NOS. 9 or 10) may be inverted relative to its normal presentation for transcription to produce antisense nucleic acid molecules. In one embodiment the antisense molecules can be used to inhibit P450RAI-3 expression and/or retinoic acid metabolism.

The antisense nucleic acid molecules of the invention or a fragment thereof, may be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed with mRNA or the native gene e.g. phosphorothioate derivatives and acridine substituted nucleotides. The antisense sequences may be produced biologically using an expression vector introduced into cells in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense sequences are produced under the control of a high efficiency regulatory region, the activity of which may be determined by the cell type into which the vector is introduced.

The invention also provides nucleic acids encoding fusion proteins comprising a novel protein of the invention and a selected protein, or a selectable marker protein (see below).

The proteins of the invention (including modifications, variations, truncations, insertions, analogs, fusion proteins, etc.) may be prepared using recombinant DNA methods. These proteins may be purified and/or isolated to various degrees using techniques known in the art. Accordingly, nucleic acid molecules of the present invention having a sequence which encodes a protein of the invention may be incorporated according to procedures known in the art into an appropriate expression vector, which ensures good expression of the protein. Possible expression vectors include but are not limited to cosmids, plasmids, or modified viruses (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), so long as the vector is compatible with the host cell used. The expression “vectors suitable for transformation of a host cell”, means that the expression vectors contain a nucleic acid molecule of the invention and regulatory sequences, selected on the basis of the host cells to be used for expression, which are operatively linked to the nucleic acid molecule. “Operatively linked” is intended to mean that the nucleic acid is linked to regulatory sequences in a manner, which allows expression of the nucleic acid.

The invention therefore contemplates a recombinant expression vector of the invention containing a nucleic acid molecule of the invention, or a fragment thereof, and the necessary regulatory sequences for the transcription and translation of the inserted protein-sequence. Suitable regulatory sequences may be derived from a variety of sources, including bacterial, fungal, or viral genes (For example, see the regulatory sequences described in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Selection of appropriate regulatory sequences is dependent on the host cell chosen, and may be readily accomplished by one of ordinary skill in the art. Examples of such regulatory sequences include: a transcriptional promoter and enhancer or RNA polymerase binding sequence, a ribosomal binding sequence, including a translation initiation signal. Additionally, depending on the host cell chosen and the vector employed, other sequences, such as an origin of replication, additional DNA restriction sites, enhancers, and sequences conferring inducibility of transcription may be incorporated into the expression vector. It will also be appreciated that the necessary regulatory sequences may be supplied by the native protein and/or its flanking regions.

The invention further provides a recombinant expression vector comprising a DNA nucleic acid molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression, by transcription of the DNA molecule, of an RNA molecule which is antisense to a nucleotide sequence comprising the nucleotides as shown in FIG. 1 or 2 (SEQ. ID. NOS. 9 or 10) or fragments thereof. Regulatory sequences operatively linked to the antisense nucleic acid can be chosen which direct the continuous expression of the antisense RNA molecule.

The recombinant expression vectors of the invention may also contain a selectable marker gene, which facilitates the selection of host cells transformed or transfected with a recombinant molecule of the invention. Examples of selectable marker genes are genes encoding a protein which confers resistance to certain drugs, such as G418 and hygromycin. Examples of other markers which can be used are: green fluorescent protein (GFP), b-galactosidase, chloramphenicol acetyltransferase, or firefly luciferase. Transcription of the selectable marker gene is monitored by changes in the concentration of the selectable marker protein such as b-galactosidase, chloramphenicol acetyltransferase, or firefly luciferase. If the selectable marker gene encodes a protein conferring antibiotic resistance such as neomycin resistance transformant cells can be selected with G418. Cells that have incorporated the selectable marker gene will survive, while the other cells die. This makes it possible to visualize and assay for expression of recombinant expression vectors of the invention and in particular to determine the effect of a mutation on expression and phenotype. It will be appreciated that selectable markers can be introduced on a separate vector from the nucleic acid of interest.

The recombinant expression vectors may also contain genes which encode a fusion moiety which provides increased expression of the recombinant protein; increased solubility of the recombinant protein; and aid in the purification of a target recombinant protein by acting as a ligand in affinity purification. For example, a proteolytic cleavage site may be added to the target recombinant protein to allow separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.

Recombinant expression vectors can be introduced into host cells to produce a transformed host cell. The term “transformed host cell” is intended to include prokaryotic and eukaryotic cells which have been transformed or transfected with a recombinant expression vector of the invention. The terms “transformed with”, “transfected with”, “transformation” and “transfection” are intended to encompass introduction of nucleic acid (e.g. a vector) into a cell by one of many possible techniques known in the art. Prokaryotic cells can be transformed with nucleic acid by, for example, electroporation or calcium chloride mediated transformation. Nucleic acid can be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co precipitation, DEAE-dextran-mediated transfection, lipofectin, electroporation or microinjection. Suitable methods for transforming and transfecting host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other such laboratory textbooks.

Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. For example, the proteins of the invention may be expressed in bacterial cells such as E. coli, insect cells (using baculovirus), yeast cells or mammalian cells. Other suitable host cells can be found in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego Calif. (1991).

The proteins of the invention may also be prepared by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis (Merrifield, 1964, J. Am. Chem. Assoc. 85:2149-2154) or synthesis in homogenous solution (Houbenweyl, 1987, Methods of Organic Chemistry, ed. E. Wansch, Vol. 15 I and II, Thieme, Stuttgart).

iii) Polynucleotide and Polypeptide Variants

“Variant” refers to a polynucleotide or polypeptide differing from the P450RAI-3 polynucleotide or polypeptide, but retaining essential properties thereof. Typically, variants are overall closely similar and, in many regions, identical to the P450RAI-3 polynucleotide or polypeptide. The invention includes homologs, analogs and isoforms of the polypeptides and as applicable polynucleoitdes of the invention. Insertions, deletions, mutations and substitutions are also intended to be encompassed within the scope of the invention.

It will be appreciated that the invention includes polynucleotides comprising nucleic acid sequences having substantial sequence homology with the sequences of FIG. 1 or 2 (SEQ. ID. NOS. 9 or 10). The term “sequences having substantial sequence homology” means those nucleic acid sequences which have slight or inconsequential sequence variations from these sequences, i.e., the sequences function in substantially the same manner to produce functionally equivalent proteins. The variations may be attributable to local mutations or structural modifications. Preferably such polucucleotides have at least 85, preferably 90 and most preferably 95% identity with the sequence of FIG. 1 or 2 (SEQ. ID. NOS. 9 or 10). However, it should be noted that the invention is not limited thereto and includes polynucletide sequence having at least 50%, 60% and 70% homology to the sequence of FIG. 1 or 2 (SEQ. ID. NOS. 9 or 10).

By a polynucleotide having a nucleotide sequence at least, for example, 90% “identical” to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the polypeptide is identical to the reference sequence except that the polynucleotide sequence may include up to ten point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the P450RAI-3 polypeptide. Therefore, to obtain a polynucleotide having a nucleotide sequence at least 90% identical to a reference nucleotide sequence, up to 10% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 10% of the total nucleotides in the reference sequence may be inserted into a reference sequence. The query sequence may be an entire nucleotide sequence of P450RAI-3 or any fragment specified as described herein.

Whether a particular nucleic acid molecule or polypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% homologous to a nucleotide sequence of the present invention can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence [a sequence of the present invention] and a subject sequence, also referred to as a global alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al., Comp. App. Biosci. 6: 237-245 (1990). In a sequence alignment, the query and the subject sequence are both DNA sequences. An RNA sequence can be compared by converting U's to T's. The result of said global sequence alignment is in percent identity. Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are: Matrix-Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization Group Length=09, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of the subject nucleotide sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence because of 5′ or 3′ deletions, not because of internal deletions, a manual correction must be made to the result. This is because the FASTDB program does not account for 5′ and 3”truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5′ or 3′ ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5′ and 3′ of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specific parameters, to arrive at a final percent identity score. This corrected score is what is used for the purposes of the present invention. Only bases outside the 5′ and 3′ bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated from the purposes of manually adjusting the percent identity score.

For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the 5′ end of the subject sequence and therefore, the FASTDB alignment does not show a match/alignment of the first 10 bases at 5′ end. The 10 unpaired bases represent 10% of the sequence [number of bases at the 5′ and 3′ ends not matched/total number of bases in the query sequence] so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%. As another example, a 90 base subject sequence may be compared with a 100 base query sequence. This time the deletion may be an internal deletion so that there are no bases on the 5′ or 3′ end of the subject sequence which are not matched/aligned with the query. In such a case, the percent identity calculated by FASTDB is not manually corrected. Only bases 5′ and 3′ of the subject sequence which are not match/aligned with the query sequence are manually corrected.

By a polypeptide having an amino acid sequence at least, for example, 90% “identical” or homologous to a query amino acid sequence of the present invention, such as P450RAI-3 (SEQ. ID. NO. 11), it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to ten amino acid alterations per each 100 amino acids of the query amino acid sequence. Therefore, to obtain a polypeptide having an amino acid sequence at least 90% identical to a query amino acid sequence, up to 10% of the amino acid residues in the subject sequence may be inserted, deleted or substituted with another amino acid. The alterations in the reference sequences may occur at the amino or carboxy terminal position of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.

Whether any particular polypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequences encoded by clone RP11-30F3 can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence [a sequence of the present invention] and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al., Comp. App. Biosci. 6: 237-245 (1990). In a sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The results of said global sequence alignment is in percent identity. Preferred parameters used in a FASTDB amino acid alignment are: Matrix-PAM 0, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. Whether a residue is match/aligned is determined by results of FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specific parameters, to arrive at a final percent identity score. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score.

For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence [number of residues at the N- and C-termini not matched/total number of residues in the query sequence] so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Only residues positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected for.

P450RAI-3 variants may contain alterations in the coding regions, non-coding regions or both. Especially preferred are polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred. Moreover, variants in which 5-10, 1-5, or 1-2 amino acids are substituted, deleted or added in any combination are also preferred. P450RAI-3 polynucleotide variants can be produced for a variety of reasons including to optimize codon expression for a particular host.

Naturally occurring P450RAI-3 variants are called “allelic variants” and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. These allelic variants can vary at either the polynucleotide and/or polypeptide level. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis. It will be appreciated that variant forms of polynucleotides of the invention which arise by alternative splicing of an mRNA corresponding to a cDNA of the invention are encompassed by the invention.

Using known methods of protein engineering and recombinant DNA technology, variants may be generated to improve or alter the characteristics of P450RAI-3 polypeptide. For example, one or more amino acids may be deleted from the N-terminus or C-terminus of the protein without substantial loss of biological function [see for example Ron et al., J. Biol. Chem. 268: 2984-2988 (1993); Dobeli et al. J. Biotechnology 7; 199-216 (1988); and Gayle et al. J. Biol. Chem. 268: 22105-22111].

If deleting one or more amino acids from the N-terminus or C-terminus of a polypeptide results in modification or loss of one or more biological functions, other biological activities may still be retained. For example, the ability of a deletion variant to induce and/or to bind antibodies which recognize the secreted form will likely be retained when less than the majority of the residues of the secreted form are removed from the N-terminus or the C-terminus. Whether a particular polypeptide lacking N- or C-terminal residues of a protein retains such immunological activities can readily be determined by routine methods described herein and know in the art.

The invention further includes P450RAI-3 polypeptide variants which show substantial biological activity. Such variants include deletions, insertions, inversions, repeats and substitutions selected according to general rules known in the art [see for example Bowie, J. U. et al., Science 247:1306-1310 (1990)].

One strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, conserved amino acids can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acids positions where substitutions have been tolerated by natural selection indicates that these positions are not critical for protein function. Therefore, positions tolerating amino acid substitutions could be modified while still maintaining biological activity of the protein.

Another strategy employs genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site directed mutagenesis or alanine-scanning mutagenesis can be used [see for example Cunningham and Wells, Science 244; 1081-1085 (1989)]. The resulting polypeptide may be tested for biological activity.

Besides conservative amino acid substitutions, this invention contemplates variants of P450RAI-3 including [1] substitutions with one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code, or [2] substitution with one or more of the amino acid residues having a substituent group, or [3] fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the polypeptide or [4] fusion of the polypeptide with additional amino acids, such as an IgGFc fusion region peptide, or a sequence facilitating purification. Such variants are deemed to be within the scope of those skilled in the art from teachings herein.

For example, P450RAI-3 polypeptide variants containing amino acid substitutions of charged amino acids with another charged or neutral amino acids my produce polypeptides with improved characteristics [see for example. Pinckard et al., Clin. Exp. Immunol. 2: 331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987); and Clevland et al., Crit. Rev. Therapeutic Drug Carrier System 10: 307-377 9!993)].

iv) Polynucleotide and Polypeptide Fragments

In the present invention “polynucleotide fragment” refers to a short polynucleotide having part of a nucleic acid sequence of FIG. 1 or 2. The short nucleotide fragments are preferably at least about 15 nt and more preferably at least about 20 nt, still more preferably at least about 30 nucleotide (nt), and even more preferably, at least about 40 nt in length. For example, a fragment “at least 20 nt is length” is intended to include 20 or more contiguous bases from the cDNA sequences of FIG. 1 or 2 (SEQ. ID. NOS. 1 or 2) or the nucleotide sequence encoding the peptide of FIG. 3 (SEQ. ID. NO. 11). The nucleotide fragments may be useful as diagnostic probes and primers. In addition, larger fragments are also useful as diagnostic probes [for example. 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750,800,850,900, 950,1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450,1500,1550, etc . . . nucleotides.] Further any of the nucleotide sequences encoding the exons shown in FIG. 5 are also intended to be included in the invention.

In one embodiment the polynucleotide fragments of the invention preferably hybridize to nucleic acid molecules of the invention (such as FIG. 1 or 2) under hybridization conditions, preferably stringent hybridization conditions. Appropriate stringency conditions which promote DNA hybridization are known to those skilled in the art, or may be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the following may be employed: 6.0× sodium chloride/sodium citrate (SSC) at about 45° C., followed by a wash of 2.0×SSC at 50° C. The stringency may be selected based on the conditions used in the wash step. For example, the salt concentration in the wash step can be selected from a high stringency of about 0.2×SSC at 50° C. In addition, the temperature in the wash step can be at high stringency conditions, such as at about 65° C.

Examples of representative polynucleotide fragments are 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351400, 401450, 451-500, 501-550, 551-600, 601-650, 651-700, 701-750, 751-800, 801-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-etc . . . to the end of the cDNA contained in FIG. 2 or the nucleotide sequences encoding any one or more of the exons 1 to 6 in FIG. 5. In a preferred embodiment the polynucleotide fragment comprises or consists of all or at least about a 15 nucleotide portion encoding SEQ. ID. NO. 8. In this context, “about” includes the particular ranges that may be larger or smaller by several nucleotides at either terminus or at both termini. Preferably, these fragments encode a polypeptide which has biological activity. More preferably, these polynucleotides can be used as probes or primers as discussed herein.

In the present invention, a “polypeptide fragment” refers to a short amino acid sequence of FIG. 3 (SEQ. ID. NO. 11) or encoded by the cDNA of FIG. 2 (SEQ. ID. NO. 10). Protein fragments may be “free-standing” or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region. For example, polypeptide fragments may include fragments from about amino acid number 1-20, 21-40, 41-60 etc. to the end of the coding region. The polypeptide fragments may be about 20, 30, 40, 50, 60, 70, 80, 90, etc. amino acids in length. “About” includes the ranges described herein and ranges larger or smaller by several amino acids, at either extremes or both extremes. The polypeptide fragments may also include anyone or more of exons 1 to 6 of FIG. 5 (SEQ. ID. NOS. 13, 15, 17, 19, 21 or 23).

Preferred polypeptide fragments include the nascent and mature forms of P450RAI-3. Furthermore, any combination of amino and carboxy terminus deletions are preferred. For example, the ability of shortened P450RAI-3 mutants to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by methods known in the art. It is not unlikely that a P450RAI-3 mutant with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as five amino acid residues may evoke an immune response.

Accordingly, the present invention further provides polypeptides having one or more residues deleted from the amino terminus of P450RAI-3. For example, the ability of the shortened P450RAI-3 mutant to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described in the art. It is not unlikely that a P450RAI-3 mutant with a large number of deleted C-terminal amino acid residues may retain some biological or immunological activities.

The invention also contemplates polypeptides having one or more amino acids deleted from both the amino and the carboxy termini of P450RAI-3 polypeptide.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences would be related to P450RAI-3 sequence and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention.

Preferred fragments are those demonstrating some biological or biochemical activity. Such fragments are those exhibiting activity similar, but not necessarily identical to P450RAI-3 polypeptide or polynucleotide. The activity may include an improved desired activity or a decreased undesired activity. Such fragments would also include, but is not necessarily limited to any polypeptide or polynucleotide fragments which are beneficial in the modulation or simulation of P450RAI-3 or P450RAI-3 expression, or in the identification or production of such agents.

v) Epitopes and Antibodies

In the present invention, “epitope” refers to P450RAI-3 or fragments having antigenic or immunogenic activity in an animal. A preferred embodiment of the present invention relates to P450RAI-3 fragment comprising an epitope, as well as the polynucleotide encoding fragment. A region of a protein molecule to which an antibody can bind is defined as an “antigenic epitope”. In contrast, an “immunogenic epitope” is defined as a part of a protein that elicits an antibody response [see for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81: 3998-4002 (1983)].

Fragments which function as epitopes may be produced by any conventional means. [see for example, Houghten, R. A., Proc. Natl. Acad. Sci. USA 82: 5131-5135 (1985)].

In the present invention, antigenic epitopes preferably contain a sequence of seven or more, more preferably at least nine, and most preferably, between about 15 to about 30 amino acids. Antigenic epitopes are useful to raise antibodies, including monoclonal antibodies, that specify binding the epitope [see for example, Wilson et al., Cell 37: 767-778 (1984); and Sutcliffe J. G. et al., science 218: 660-666].

Similarly, immunogenic epitopes can be used to induce T cells according to methods well known in the art [see for example, Chow, M. et al. Proc. Natl. Acad. Sci. USA 82: 910-914; and Bittle, F. J. et al. J. Gen. Virol. 66: 2347-2354 (1985)]. The immunogenic epitope may be presented together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse) or, if it is long enough (at least about 25 amino acids, without a carrier. However, immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide such as Western blotting.

As used herein, the term “antibody” [Ab] or “monoclonal antibody” [mAb] is meant to include intact molecules as well as antibody fragments [for example Fab and F(ab′)2 fragments] which are capable of specifically binding proteins. Such fragments lack the Fc fragment of intact antibody and are typically produced by proteolytic cleavage using enzymes such as papin (to produce Fab fragments) or pepsin (to produce F(ab′) fragments). Fab and F(ab′)2 fragments clear more rapidly from the circulation and may have less non-specific tissue binding than an intact antibody [see for example Wahl et al., J. Nucl. Med. 24: 316-325 (1983)]. Thus these fragments are preferred, as are the products of a Fab or other immunoglobulin expression library. This invention includes chimeric, single chain and humanized antibodies. In addition, target protein-binding fragments can be produced through the application of recombinant DNA technology or through synthetic chemistry.

These methodologies are known in the art. For further references, see examples including Current Protocols in Immunology, John Wiley & Sons, New York; Kennett, R. et al, eds., Monoclonal Antibodies, Hybridoma: A New Dimension in Biological Analysis, Plenum Press, New York (1980) and Campbell, A., “Monoclonal Antibody Technology” in Burden, R., et al., eds., Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13, Elsevere, Amsterdam (1984).

Antibodies generated against a target epitope can be obtained by direct injection of the epitope or polypeptide into an animal or by administrating the polypeptides to an animal, preferably a nonhuman. Such an antibody will then bind the polypeptide itself. With this method, a sequence encoding only a fragment of the polypeptide can be used to generate antibodies binding the whole native polypeptide. Such antibodies can be used to isolate the polypeptide encoding the polypeptide from an expression library using the method described herein.

For the preparation of monoclonal antibodies, any technique that provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique [Kohler and Milstein, Nature 256: 495-497 (1975)], the trioma technique, the human B-cell hybridoma technique [Kozbor et al., Immunol. Today 4: 72 (1983)], and the EBV-hybridoma technique to produce human monoclonal antibodies [Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. p 77-96 (1985)].

Techniques described for the production of a single chain antibodies [U.S. Pat. No. 4,946,778] can be adapted to produce single chain antibodies to immunogenic polypeptide products of interest.

The antibodies useful in the present invention may be prepared by any of a variety of methods known in the art. For example, cells expressing the target protein or an antigenic fragment thereof can be administered to an animal in order to induce the introduction of sera containing polyclonal antibodies. In another method, a preparation of target protein is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.

In a preferred method, antibodies used in the present invention are monoclonal antibodies [or target protein-binding fragments thereof]. Such monoclonal antibodies can be prepared using hybridoma technology known in the art. In general, such procedures involve immunizing an animal [preferably a mouse] with a target protein antigen or, preferably, with a target protein-expressing cell. Suitable cells can be recognized by their capacity to bind an anti-target protein antibody. Such cells may be cultured in any suitable tissue culture medium. The splenocytes of immunized mice are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention. Preferably SP20 myeloma cell line is used which is available from American Type Culture Collection, Mannassas, Va. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium followed by cloning out by limited dilution as described in the art [see for example, Wands et al., Gastroenterology 80: 225-232 (1981)]. The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the target protein antigen.

Alternatively, additional antibodies capable of binding to the target protein antigen may be produced in a two-step procedure through the use of anti-idiotypic antibodies. Such a method makes use of the fact that antibodies are themselves antigens, and that, therefore, it is possible to obtain an antibody which binds to second antibody. With this method, target-protein specific antibodies are used to immunize an animal, preferably a mouse. The splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce an antibody whose ability to bind to the target protein-specific antibody can be clocked by the target antigen. Such antibodies comprise anti-idiotypic antibodies to the target protein-specific antibody and can be used to immunize an animal to induce formation of further target protein-specific antibodies.

Suitable labels for the target protein-specific antibodies of the present invention are provided below. Examples of suitable enzyme labels include malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast-alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholine esterase.

Examples of suitable radioactive labels include ³H, ¹¹¹In, ¹²⁵I, ¹³¹I, ³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ⁵⁷To, ⁵⁸Co, ⁵⁹Fe, ⁷⁵Se, ¹⁵²Eu, ⁹⁰Y, ⁶⁷Cu, ²¹⁷Ci, ²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd etc. Examples of suitable non-radioactive isotopic labels include ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Tr and ⁵⁶Fe.

Examples of suitable fluorescent labels include an ¹⁵²Eu label, a fluorescein label, an isothiocyanate label, a rhodamine label, a phycoerythrin label, a phycocyanin label, an allophycocyanin label, an o-phthaldehyde label and a fluorescamine label.

Examples of chemiluminescent labels include a luminal label, an isoluminal label, an aromatic acridinium ester label, an imidazole label, an acridinium salt label, an oxalate ester label, a luciferin label, a luciferase label and an aequorin label.

Examples of nuclear magnetic resonance contrasting agents include heavy metal nuclei such as Gd, Mn and iron.

Typical techniques for binding the above-described labels to antibodies are provided by Kennedy et al., Cin. Chem. Acta. 70;1-31 (1976) and Schurs et al., Clin. Cem. Acta 82: 1-40 (1970). Coupling techniques mentioned in the latter are gluteraldehyde method, the periodate method, the dimaleimide method, the m-maleimidobenzyl-N-hydroxy-succinimide ester method, all of which methods are incorporated by reference herein.

For in vivo use of antibodies in humans, it may be preferable to use “humanized” chimeric monoclonal antibodies. Such antibodies can be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described herein. Methods for producing chimeric antibodies are known in the art. (For example, Morrison, Science 229:1202 (1985); Oi et al. Bio Techniques 4:214 91986); cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne et al, Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985).)

vi) Disease States Diagnosis and Prognosis

It is believed that certain conditions may cause mammals to express significantly altered levels of P450RAI-3 protein and mRNA levels encoding P450RAI-3 protein when compared to a corresponding “standard” mammal i.e., a mammal of the same species not having the condition. Cytochrome P450s have been associated with a number of pathways and have been implicated in a number of medical conditions. In addition to conditions which may be associated with cytochrome P450s in general, P450RAI-3 has elevated expression in the adrenal gland. This coupled with the degree of homology with the other retinoic acid metabolizing cytochrome P450s (P450RAI-1 and -2) suggest a role in cellular differentiation of carcinogenic, tumor and embryonic cell lines. Thus the polypeptides and polynucleotides of this invention or modulators thereof may be useful in diagnosing and treating conditions related to cellular differentiation, such as cancer or developmental disorders. It may also be useful in treating conditions such as psoriasis, high blood pressure, conditions related to sterol or hormone disorders, to name a few and other conditions of the adrenal glands noted herein.

Further, P450RAI-3 or the polypeptides, polynucleotides of this invention and/or modulators of P450RAI-3 activity may be useful in treating disorders or conditions involving the vitamin A or retinoic acid metabolic pathway, such as those noted herein.

Where a diagnosis has already been made according to conventional methods, the present invention is useful as a prognostic indicator whereby patients exhibiting altered P450RAI-3 gene expression will experience a worse or better clinical outcome relative to patients expressing the gene at a normal level.

By “assaying the expression of the gene encoding the P450RAI-3 polypeptide” is intended qualitatively and quantitatively measuring or estimating the level of P450RAI-3 protein or the level of the mRNA encoding the P450RAI-3 protein in a first biological sample either directly [e.g., by determining or estimating absolute protein level or mRNA level] or relatively [e.g. by comparing to the P450RAI-3 protein level or mRNA level in a second biological sample].

Preferably, the P450RAI-3 protein level or mRNA level in the first biological sample is measured or estimated and compared to a standard P450RAI-3 protein level or RNA level, the standard being taken from a second biological sample obtained from an individual not having the condition. As will be appreciated in the art, once a standard P450RAI-3 protein level or mRNA level is known, it can be used repeatedly as a standard for comparison.

By “biological sample” is intended any biological sample obtained from an individual, cell line, tissue culture or other source which contains CYP26C or mRNA. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.

Preferred mammals include monkeys, apes, cats, dogs, cows, pigs, horses, rabbits, mice, and humans. Particularly preferred are humans.

Total cellular RNA can be isolated by methods well known in the art [see for example, Chmczynski and ZSacchi, Anal. Biochem. 162: 156-159 (1987)]. Levels of mRNA encoding the P450RAI-3 protein are then assayed using an appropriate method. These include Northern blot analysis [Harada et al., Cell 63: 303-312 (1990)], S1 nuclease mapping [Fujita et al., Cell 49: 357-367 (1987)], the polymerase chain reaction [PCR], reverse transcription in combination with the polymerase chain reaction [RT-PCR] [Mariko et al., Technique 2: 295-301 (1990)] and reverse transcription in combination with the ligase chain reaction [RT-LCR].

Assaying P450RAI-3 protein levels in a biological sample can occur using antibody-based techniques. For example, P450RAI-3 protein expression in tissue can be studied with classical immunohistological methods known in the art [for example, Jalkanen, M., et al., J. Cell Biol. 105: 3087-3096 (1987)].

Other antibody-based methods used for detecting P450RAI-3 protein gene expression including immunoassays, such as enzyme linked immunosorbent assay [ELISA] and the radioimmunoassay [RIA].

Suitable labels are known in the art and include enzyme labels, such as, glucose oxidase, and radioisotope, such as iodine [¹²⁵I, ¹²¹I], carbon [¹⁴C], sulfer [³⁵S], tritium [³H], indium [¹¹²In] and technetium [^(99m)Tc] and fluorescent labels, such as fluorescein and rhodamine and biotin.

Antibody labels or markers for in vivo imaging of protein include those detectable by X-radiography, NMR or ESR. For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into antibody by labelling of nutrients for the relevant hybridoma.

A protein-specific antibody or antibody fragment which has been labelled an appropriate detectable imaging moiety, such as a radioisotope [for example, ¹³¹I, ¹¹²¹n, ^(99m)TC], a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced [for example, parenterally, subcutaneously, or intraperitoneally] into mammals. It will be understood in the art that the size of the subject and the imaging system used will determine the quality of imaging moiety needed to produce diagnostic images. In vivo tumour imaging is described in S. W. Burchiel et al., “Immunopharmackinetics of Radiolabeled Antibodies and Their Fragments” [see also, Chapter 13 in Tumour Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982)].

vii) Fusion Proteins

Any P450RAI-3 polypeptide (any polypeptide of the invention—hybrid, variations, fragments, etc.) may be used to generate fusion proteins. For example, the P450RAI-3 polypeptide, when fused to a second polypeptide, can be used as an antigenic tag. Antibodies raised against the P450RAI-3 polypeptide can be used to indirectly detect the second protein by binding to the P450RAI-3 polypeptide. Examples of domains that can be fused to P450RAI-3 include heterologous functional regions. The fusion does not necessarily need to be direct, but may occur through linker sequences.

In certain preferred embodiments, P450RAI-3 fusion polypeptides may be constructed which include additional N-terminal and/or C-terminal amino acid residues. In particular, any N-terminally or C-terminally deleted P450RAI-3 polypeptide disclosed herein may be altered by inclusion of additional amino acid residues at the N-terminus to produce a P450RAI-3 fusion polypeptide, In addition, P450RAI-3 fusion polypeptides are contemplated which include additional N-terminal and/or C-terminal amino acid residues fused to a P450RAI-3 polypeptides comprising any combination of N- and C-terminal deletions set forth above.

In addition, fusion proteins may be engineered to improve characteristics of the P450RAI-3 polypeptide. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the P450RAI-3 polypeptide to improve stability and persistence during purification from the host cell or subsequent handling and storage. Furthermore, peptide moieties may be added to the P450RAI-3 polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the P450RAI-3 polypeptide. The addition of peptide moieties to facilitate handling of polypeptides are familiar and routine techniques in the art.

P450RAI-3 polypeptides, including fragments, and specifically epitopes, can be combined with parts of the constant domain of immunoglobulins [IgG], resulting in chimeric polypeptides. These proteins facilitate purification and show an increased half-life in vivo. One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. (EP A 394, 827; Traunecker et al., Nature 331: 84-86 (1988). Fusion proteins having disulphide-linked dimeric structures [due to the IgG] can also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone [see for example, Fountoulakis et al., J. Biochem. 270: 3958-3964 (1995)].

Similarly, EP-A-O 469 533 (Canadian counterpart 2045869) disclosed fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties. (EP-A 0232 262). Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In addition, in drug discovery, human proteins, such as hIL-5, have been fused with FC portion for the purpose of high-throughout screening assays to identify antagonists of hIL-5 [see for example, Bennet, D. et al., J. Molecular Recognition 8: 52-58 (1995); K. Johanson et al., J. Biol. Chem. 270: 9459-9471 (1995)].

Moreover, the P450RAI-3 polypeptides of the invention can be fused to other proteins, e.g. NADPH cytochrome P450 reductase, NADPH ferredoxin reductase, other flavoproteins or ferrodixins or other proteins or co-factors which may function as a cytochrome P450RAI-3 reductase or facilitate such an activity to create a multiprotein fusion complex. Such a multiprotein fusion complex may function as an enzymatically active covalently linked P450RAI-3-reductase complex. A multiprotein complex can be synthesized by the means of chemical crosslinking or assembled via novel intramolecular interactions, e.g., by the use of specific antibodies stabilizing the complex.

P450RAI-3 polypeptide can be fused to hydrophilic molecules, including but not limited to polyethylene glycol and modified oligosaccharide and polysaccharides, whereby the hydrophobic moieties are used to stabilize P450RAI-3 interactions with other proteins, natural membranes, or artificial membranes, or to create new interactions with other proteins, natural membranes or artificial membranes. Fusion of the P450RAI-3 polypeptide to hydrophilic molecules can also be used to change its solubility.

P450RAI-3 polypeptide variants which contain non-standard amino acids or additional chemical modifications which have use in purification, stabilization or identification of the resulting modified P450RAI-3 protein, or influence its other properties such as enzymatic activity or interaction with other proteins, membranes, solid supports or chromatographic resin are contemplated. This includes, but is not limited to, biotinylated derivatives or fusions of P450RAI-3 polypeptides.

Also, modification of the P450RAI-3 polynucleotide sequence include those where relevant regions of the P450RAI-3 gene or polypeptide are inserted into another gene sequence to create a chimeric protein with a desired activity (enzymatic or otherwise). Such chimeric proteins can be obtained by, for example, replacing regions of other cytochrome P450 genes or polypeptides with a relevant P450RAI-3 regions whereby such a modification confers a new functional property to the resulting chimeric protein, including but not limited to new specificity, changed enzymatic kinetics, new or changed interactions with reductase or other relevant molecules or membranes, changed solubility and changed stability.

Moreover, the P450RAI-3 polypeptide can be used to marker sequences, such as a peptide which facilitates purification of P450RAI-3. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide [His-tag], such as the tag provided in a pQE vector [QIAGEN, In., 9259 Eton Avenue, Chatsworth, Calif., 91311], among others, many of which are commercially available. [for example see Gentz et al., Proc. Natl. Acad. Sci. USA 86: 821-824 (1989)], for instance, hexa-histidine provides for convenient purification of the fusion protein. Another peptide tag useful for purification, the “HA” tag, corresponding to an epitope derived from the influenza haemagglutonin protein [see for example, Wilson et al., Cell 37: 767 (1984)].

Any of these fusions can be engineered using the P450RAI-3 polynucleotides or the P450RAI-3 polypeptides of this invention.

viii) Vectors, Host Cells and Protein Production

The present invention also relates to vectors containing the polynucleotides of the invention, preferably the polynucleotide encoding P450RAI-3, host cells and to the production of the polypeptides of the invention, preferably the P450RAI-3 polypeptide, by recombinant techniques. For example, the vector may be a phage, plasmid, viral, or retroviral. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.

Introduction of constructs into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology (1986).

P450RAI-3 can be recovered and purified from recombinant cell cultures by methods well-known in the art including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography [“HPLC”] may be employed for purification.

In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary and immortalized host cells of vertebrate origin, particularly mammalia, origin, that have been engineered to delete or replace endogenous genetic material and/or to include genetic material [e.g. heterologous polynucleotide sequences] that is operably associated with P450RAI-3 polynucleotide of the invention, and which activates, alters, and/or amplifies endogenous P450RAI-3 polynucleotides. For example, techniques known in the art may be used to operably associate heterologous control regions [e.g. promoter and/or enhancer] and endogenous P450RAI-3 polynucleotide sequences via homologous recombinations [see for example Koller et al., Proc. Natl. Acad. Sci. 86: 8932-8935 (1989); and Zijilstra et al., Nature 342: 435438 (1989)].

ix) Uses of P450RAI-3 Polynucleotide

The P450RAI-3 polynucleotides referred to herein can be used in numerous ways as agents. The following describes some examples using techniques know in the art.

There exists an ongoing need to identify new chromosome markers, since few chromosome marking reagents, based on actual sequence data [repeat polymorphisms], are presently available.

Briefly, sequences can be mapped to chromosomes by PCR primers [preferably 5-25 bp] from the sequence shown in FIG. 1 or 2. Primers can be selected using computer analysis so that primers do not span more than one predicted exon in the genomic DNA. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human P450RAI-3 gene corresponding to the given sequence (preferably the sequence of FIG. 2) will yield an amplified fragment.

Similarly, somatic hybrids provide a rapid method of PCR mapping of the polynucleotides to particular chromosomes. Three or more clones can be assigned per day using a single thermal cycle. Moreover, sublocalization of the P450RAI-3 polynucleotide may be achieved with panels of specific chromosome fragments. Other gene mapping strategies that can be used include in situ hybridization, prescreening with labelled flow-sorted chromosomes and preselection by hybridization to construct chromosomes specific-cDNA libraries.

Precise chromosomal location of P450RAI-3 polynucleotides can also be achieved using fluorescent in situ hybridization [FISH] of a metaphase chromosomal spread. This technique uses polynucleotides as short as 500 or 600 bases; however, polynucleotides 2,000-4,000 bp are preferred [see for example. Verma et al., “Human Chromosomes: a Manual of Basic Techniques”, Pergamon Press, New York (1988)].

For chromosome mapping, the P450RAI-3 polynucleotide can be used individually [to mark a single chromosome or a single site on that chromosome] or in panel [for marking multiple sites and/or multiple chromosomes]. Preferred polynucleotides corresponding to the noncoding regions of the cDNAs becomes the coding sequences are more likely conserved within gene families, thus increasing the chance of cross hybridization during chromosomal mapping.

Once a polynucleotide has been mapped to a precise chromosomal location, the physical position of the polynucleotide can be used in linkage analysis. Linkage analysis establishes coinheritance between a chromosomal location and presentation of a particular disease. Assuming 1 megabase mapping resolution and one gene per 20 kb, a cDNA precisely localized to a chromosomal region associated with the disease could be one of 50-500 potential causative genes.

Therefore, once coinheritance is established, differences in the P450RAI-3 polynucleotide and the corresponding gene between affected and unaffected individuals can be examined. First, visible structural alterations in the chromosomes, such as deletions or translocations, are examined in chromosomes spreads or by PCR. If no structural alterations exist, the presence of point mutations is ascertained. Mutations observed in some or all affected individuals, but not in normal individuals, indicates that the mutation may cause the disease. However, complete sequencing of the P450RAI-3 polynucleotide and the corresponding gene from several normal individuals is required to distinguish the mutation from a polymorphism. If a new polymorphism is identified, this polymorphic polypeptide can be used for further linkage analysis. The presence of a polymorphism can also be indicative of a disease or a predisposition to a disease. Therefore, a method of diagnosis of a P450RAI-3-related condition or predisposition to a P450RAI-3 relatedcondition, by identifying a polymorphism in P450RAI-3 gene, is also contemplated by this invention. In addition, a diagnostic kit for identification of polymorphisms in the P450RAI-3 gene by screening the P450RAI-3 gene from human for polymorphisms is also an embodiment of the present invention.

Furthermore, increased or decreased expression of the gene in affected individuals as compared to unaffected individuals can be assessed using P450RAI-3 polynucleotides. Any of these alterations [altered expression, chromosomal rearrangement or mutation] can be used as a diagnostic or prognostic marker.

In addition, P450RAI-3 polynucleotide can be used to control gene expression through triple helix formation or antisense DNA or RNA. Both methods rely on binding of the polypeptide to DNA or RNA. For these techniques, preferred polynucleotides are usually 20 to 40 bases in length and complementary to either the region of the gene involved in transcription or to the mRNA itself [see for example, Dervan et al., Science 251: 1360 (1991); Okano, J. Neurochem. 56; 560 (1991)]. Triple helix formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. Both techniques are effective in model systems, and the information disclosed herein can be used to design antisense or triple helix polynucleotide in an effort to treat disease.

P450RAI-3 polynucleotides are also useful in gene therapy. One goal of gene therapy is to insert a normal gene into an organism having a defective gene, in an effort to correct the gene defect. P450RAI-3 offers a means of targeting such genetic defects in a highly accurate manner. Thus, for example, cells removed from a patient can be engineered with a P450RAI-3 polynucleotide [DNA or RNA] encoding a P450RAI-3 polypeptide ex vivo, with the engineered cells then being infused back into a patient to be treated with the polypeptide. Such methods are well-known in the art. For example, cells can be engineered by procedures known in the art by use of a retroviral particle containing RNA encoding P450RAI-3.

Another goal of gene therapy is to insert a new gene that was not present in the host genome, thereby producing a new trait in the host cell.

The P450RAI-3 polynucleotides are also useful for identifying individuals from minute biological samples. The United States military, for example, is considering the use of restriction fragment length polymorphism [RFLP] for identification of person. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes and probed on a Southern blot to yield unique bands for identifying personnel. The P450RAI-3 polynucleotides can be used as additional DNA markers for RFLP.

The P450RAI-3 polynucleotides can also be used as an alternative to RFLP, by determining the actual base-by-base DNA sequence of selected portions of an individual's genome. These sequences can be used to prepare PCR primers for amplifying and isolating such selected DNA, which can then be sequenced. Using this technique, individuals can be identified because each individual will have a unique set of DNA sequences.

Forensic biology also benefits from using DNA-based identification techniques as described herein. DNA sequences taken from very small biological samples such as tissues e.g., hair, or skin or body fluids such as blood or saliva can be amplified using PCR [see for example Erlich, H. PCR Technology, Freeman and Co. (1992)]. Similarly, P450RAI-3 polynucleotide can be used as polymorphic markers for forensic purposes.

The invention provides a diagnostic method of a disorder, which involves: [1] assaying P450RAI-3 gene expression level in a biological sample from the individual, such as a tissue or cell sample of an individual; [2] comparing the P450RAI-3 gene expression level with a standard P450RAI-3 gene expression level, whereby an increase or decrease in the assayed P450RAI-3 gene expression level compared to the standard expression level is indicative of the disorder.

In the very least, the P450RAI-3 polynucleotide can be used as a molecular weight marker on Southern gels, as diagnostic probes for the presence of a specific mRNA in a cell type, as a probe to “subtract-out” known sequences in the process of discovering novel polynucleotides, for selecting and making oligomers for attachment to a “gene chip” or other support, or raise anti-DNA antibodies using DNA immunization techniques and as an antigen to elicit an immune response.

x) Uses of P450RAI-3 Polypeptides

P450RAI-3 polypeptide can be used in a number of ways including the following examples.

P450RAI-3 polypeptide can be used to assay protein levels in a biological sample using antibody-based techniques. For example, protein expression in tissue can be studied with classical immunohistological techniques [see for example, Jalkanen, M. et al., J. Cell Biol. 105: 3087-3096 (1987)]. Other antibody-based methods used for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase, and radioisotopes, such as iodine [¹²⁵I, ¹²¹I], carbon [¹⁴C], sulfer [³⁵S], tritium [³H], indium [¹¹²In] and technetium [^(99m)Tc] and fluorescent labels, such as fluorescein and rhodamine and biotin.

Moreover, the P450RAI-3 polypeptides of the invention can be used to treat disease. For example, patients can be administered P450RAI-3 polypeptides in an effort to replace absent or decreased levels of the P450RAI-3 polypeptide, to supplement absent or decreased levels of a different polypeptide or molecule, to inhibit the activity of a polypeptide to activate the activity of a polypeptide to reduce the activity of a membrane bound receptor by competing with it for free ligand, or to bring about a desired response.

Antibodies directed to P450RAI-3 polypeptide may be used to treat disease. As described in detail in the “Epitopes and Antibodies” section herein the polypeptides of the present invention can be used to raise polyclonal and monoclonal antibodies, which are useful in assays for detecting P450RAI-3 protein expression from a recombinant cell, as a way of assessing transformation of the host cell, or as antagonists capable of inhibiting P450RAI-3 protein function. Similarly, administration of an antibody can activate the polypeptide, such as by binding to a polypeptide bound to a membrane (receptor). Further, such polypeptides can be used, in the yeast two-hybrid system to “capture” P450RAI-3 protein binding proteins which are also candidate agonist and antagonist according to the present invention. The yeast two hybrid system is described in Fields and Song, Nature 340:245-246 (1989).

Small molecules that are specific substrates (such as ATRA or 9-cis RA) or metabolites of P450RAI-3 protein can also be used in the diagnosis or analysis of disease state involving P450RAI-3 or to monitor progress of therapy.

P450RAI-3 or its derivatives, P450RAI-3 fusions, complexes and chimeric proteins can also be used in the analysis of individual chemicals or complex mixtures of chemicals including, but not limited to, the screening for improved or changed small molecules. These molecules may have use in development of new therapeutic agents or new diagnostic methods for P450RAI-3-related conditions.

P450RAI-3 or its derivatives, P450RAI-3 fusions, complexes and chimeric proteins in an isolated state or as a part of complex mixtures can also be used to synthesize or modify small molecules. These molecules can in turn be used as therapeutic or diagnostic agents. Furthermore, these molecules can be used in the development of additional new molecules for therapeutic or diagnostic use.

P450RAI-3 or its derivatives, P450RAI-3 homologs, chimeras and protein fusions can be expressed in natural host cells or organisms, or in experimentally created cells or organisms for the purpose of producing, analyzing or modifying therapeutically and diagnostically important small molecules.

P450RAI-3 or its derivatives and P450RAI-3 fusions can be expressed in cells or organisms to modify the normal or diseased function and state of such hosts. In particular, this encompasses, but is not limited to, the use of P450RAI-3 polypeptides and derivatives for gene-therapy of humans or animals. P450RAI-3 polypeptides can also be used in experimental animals to reproduce physiological states, which are useful in the study and analysis of human disease, health or development.

P450RAI-3 polypeptides or derivatives and P450RAI-3 fusions can be expressed in natural host cells or organisms or in experimentally created cells or organisms and used in the extraction, conversion, localization or bioremediation of small molecules in natural or artificial environments. This use includes, but is not limited to, the removal or neutralization of environmental or industrial pollutants by cultivating transgenic or genetically modified plants or microorganisms in water or soil, or by assembling so-called bioreactors that host such organisms.

At the very least, P450RAI-3 polypeptide may be used as a molecular weight marker on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art.

xi) Heme-binding, Oxygen-binding and Detoxification

All cytochrome P450s are heme-binding proteins that contain the putative family signature, F[XX]G[XXX]C[X]G [X=any residue; conserved residues are in bold].

Heme-binding proteins, such as the cytochromes P450s, play an important role in the detoxification of toxic substances or xenobiotics. For example, toxic substances can be detoxified by oxidation. Cytochrome P450s can function as oxidative enzymes to detoxify toxic substances, such as phenobarbital, codeine and morphine. The capacity of cytochrome P450s to bind oxygen depends on the presence of a heme group and the oxygen-binding domain. Thus, the ability of P450s to bind heme and molecular oxygen enables them to detoxify toxic substances by oxidation.

Thus P450RAI-3 polypeptides are also useful as oxidative enzymes to detoxify toxic substances or xenobiotics, such as phenobarbital, codeine and morphine.

xii) Antagonist, Agonist and Antisense Methods

This invention further provides methods for screening compounds to identify agonists and antagonists to the P450RAI-3 polypeptides of the present invention.

Examples of potential P450RAI-3 agonists could include P450RAI-3 it self or biologically active fragment thereof, a variant of P450RAI-3 or biologically active fragment thereof, a nucleic acid construct encoding any of the peptide agonists, or nucleic acid, a drug or small molecule that can enhance P450RAI-3 expression or activity.

Examples of potential P450RAI-3 antagonists include antibodies, drugs, small molecules or in some cases, oligonucleotides, which bind to the polypeptides.

Antisense constructs prepared using antisense technology are also potential antagonists. Therefore, the present invention is further directed to inhibiting P450RAI-3 in vivo by the use of antisense technology. Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5′ coding portion of the polynucleotide sequence, which encodes for the [mature] polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the polypeptides [for example, antisense-Okano, J. Neurochem. 56: 560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CPR Press, Boca Raton, Fla. (1988)]. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription [triple-helix, see Lee et al., Nucl. Acids Res. 6: 3073 (1979); Cooney et al., Science 241: 456 (1988); and Dervan et al., Science 251: 1360 (1991)], thereby preventing transcription and the production of the P450RAI-3 polypeptides.

Another potential P450RAI-3 antagonist is a peptide derivative of the polypeptides which are naturally or synthetically modified analogs of the polypeptides that have lost biological function yet still recognize P450RAI-3 substrate(s). Examples of peptide derivatives include, but are not limited to, small peptides or peptide-like molecules.

The antagonist may be employed to treat disorders which are either P450RAI-3-induced or enhanced or modulated, for example, vitamin A or retinoic acid metabolic disorders.

Instead of inhibiting P450RAI-3 activity at the nucleic acid level, P450RAI-3 activity can be directly inhibited by binding to an agent, such as a suitable small molecule or drug. The present invention thus includes a method of screening drugs for their effect on activity [i.e., as a modulator (e.g. agonist or antagonist), preferably an inhibitor] of P450RAI-3 polypeptide. In particular, modulators of P450RAI-3 activity, such as drugs or peptides or other chemical compounds or molecules, can be identified in a biological assay by expressing P450RAI-3 in a cell, adding a substrate (for example 9-cis RA or ATRA) and detecting activity of P450RAI-3 polypeptide on the substrate in the presence, and optionally for a control in the absence of, the potential modulator. The assay can also be done in microsomes that comprise P450RAI-3 or other environments where P450RAI-3 is present in addition to cofactors that may be necessary for its activity such as in one embodiment NADPH cytochrome P450 reductase and/or a flavoprotein and/or NADPH. Thus, the P450RAI-3 protein can be exposed to a prospective inhibitor or modulating drug and the effect on protein activity can be determined. Prospective drugs can be tested for inhibition of the activity of other P450 cytochromes, which are desired not to be inhibited. In this way, drugs that are selectively inhibit P450RAI-3 over other P450s can be identified. Uses of the P450RAI-3 modulators identified by the assays of the invention are also encompassed within the scope of the present invention.

xiii)—Other Methods of the Invention

The methods of the invention also include a method of conducting a drug discovery and pharmaceutical business comprising:

-   -   (a) providing one or more assay systems for identifying agents         by their ability to modulate P450RAI-3 activity or expression or         retinoic acid metabolism;     -   (b) conducting therapeutic profiling of agents identified in         step (a), or further analogs thereof, for efficacy and toxicity         in animals; and     -   (c) formulating a pharmaceutical preparation including one or         more agents identified in step (b) as having an acceptable         therapeutic profile.

The method may further include a step of establishing a distribution system for distributing the pharmaceutical preparation for sale. The method can further include establishing a sales group for marketing the pharmaceutical preparation.

The present invention also provides a method of conducting a target discovery business comprising:

-   -   (a) providing one or more assay systems for identifying agents         by their ability to modulate P450RAI-3 or retinoic acid         metabolism;     -   (b) (optionally) conducting therapeutic profiling of agents         identified in step (a) for efficacy and toxicity in animals; and     -   (c) licensing, to a third party, the rights for further drug         development and/or sales for agents identified in step (a), or         analogs thereof.

xiv) Pharmaceutical Compositions

P450RAI-3 may play a role in a number of diseases or medical conditions. A “P450RAI-3 related condition” as used herein is one wherein P450RAI-3 expression or activity (whether over expression or activity, under expression or activity, or modified expression or activity) is characteristic of the disorder and/or a condition where modifying P450RAI-3 expression or activity can assist in treating the condition.

Such disorders may include but are not limited to those associated with retinoic acid expression or activity, where expression of P450RAI-3 results in oxidation of retinoic acid. As such, expression of P450RAI-3 can decrease retinoic acid levels, where such decrease may or may not be desired. P450RAI-3 may also play a role in the activity of the adrenal gland. As such, modulating P450RAI-3 expression or activity can be effective in treating conditions of the adrenal gland. Examples of such adrenal glands conditions were noted above, in the background of the invention section.

However, P450RAI-3 is not limited to being associated with such conditions only. In particular P450RAI-3 may play a more general role in cell differentiation disorders, such as cancer.

The invention comprises methods for modulating or simulating P450RAI-3 activity or P450RAI-3 expression, preferably for treating or preventing a P450RAI-3 related condition. The invention further comprises use of the modulating (any change or controlling effect on P450RAI-3 activity or expression, including administration of P450RAI-3 itself) or simulating agents disclosed herein for the preparation of a medicament for treating or preventing a condition associated with P450RAI-3 expression or activity. In another embodiment the invention provides a use of the modulating or simulating agents for the treatment or prevention of a P450RAI-3 related condition.

Accordingly, the present invention provides a method of treating or preventing a disease associated with P450RAI-3 expression or activity comprising administering an agent that modulates or simulates P450RAI-3 expression or activity to an animal in need thereof.

In one embodiment, such agents stimulate or simulate P450RAI-3 activity. Examples of agents that activate or simulate P450RAI-3 activity would include without limitations, P450RAI-3, the gene encoding for P450RAI-3 with suitable promoters, such promoters preferably being tissue specific promoters and therapeutically (or functionally) effective fragments of the nucleic acid and amino acid sequences of the invention. Further, such agent may include agonists of P450RAI-3, such as small molecules or drugs identified to have such effect.

In another embodiment, preferably theP450RAI-3, if administered is solubilized. In another embodiment, the P450RAI-3 polypeptide of the invention can be co-administered preferably with co-factors such as with the suitable NADPH cytochrome P450 reductase and preferably a flavoprotein. In another embodiment NADPH could also be administered. In another embodiment, the P450RAI-3 polypeptide of the invention can be co-administered with the substrate, retinoic acid (e.g. ATRA, 9 cis RA)e.g., where increased levels of a metabolite of retinoic acid are desired. The substrate and co-factors could both be administered with the P450RAI-3 polypeptide, or can potentially be effective alone or together.

Further, there may be diseases or conditions in which inhibition of P450RAI-3 may be required, such as in the case where retinoic acid levels or activity is to be maintained or increased. Accordingly, the invention provides a method for treating or preventing a disease or condition associated with P450RAI-3 expression or activity, (either any expression or activity or elevated expression or activity) by administering to a patient in need thereof an agent which inhibits or suppresses P450RAI-3 expression or activity.

Examples of agents that inhibit P450RAI-3 include antisense nucleic acid molecules to P450RAI-3 or fragments thereof, antibodies, antagonists, and transdominant inhibitors, as described herein.

Agents that modulate P450RAI-3 expression or activity (either alone or with another agent, such as retinoic acid, as explained above) can be formulated into pharmaceutical compositions for administration to subjects in a biologically compatible form suitable for administration in vivo. Preferably the modulator of P450RAI-3 is an inhibitor of P450RAI-3 and it is administered either alone or together with a P450RAI-3 substrate such as ATRA or 9 cis RA. Such a treatment can assist in maintaining retinoic acid levels when desired, such as in the treatment of cancer where patients may develop resistance to RA treatment, as noted above. As used herein “biologically compatible form suitable for administration in vivo” means a form of the substance to be administered in which therapeutic effects outweigh any toxic effects. The substances may be administered to animals in need thereof. Animals, as used herein refers to any animal susceptible to a P450RAI-3 related condition preferably dogs, cats, mice, horses and humans.

The pharmaceutical composition will be formulated and doses in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient (and potential side effects), the site of delivery, the method of administration, the scheduling of administration, and other factors known to a practitioner. Administration of an “effective amount” of pharmaceutical compositions of the present invention is defined as an amount of the pharmaceutical composition, at dosages and for periods of time necessary to achieve the desired result. For example, a therapeutically active amount of a substance may vary according to factors such as disease state, age, sex, and weight of the recipient, and the ability of the substance to elicit a desired response in the recipient. Dosage regime may be adjusted to provide an optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. Subject to therapeutic discretion, preferably dosages of administration of active compound (modulator of P450RAI-3 expression or activity, either alone or with another compound, such as a retinoic acid substrate) will be in the range of about 1 μg/kg/day to 10 mg/kg/day of patient body weight and most preferably at least 0.01 mg/kg/day, and most preferably for humans between about 0.01 and 1 mg/kg/day.

An active substance may be administered in a convenient manner such as by injection (subcutaneous, intravenous, topical, intratumoral etc.), oral administration, inhalation, transdermal application, or rectal administration. Depending on the route of administration, the active substance may be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound, prior to reaching the desired site of delivery. It can also be formulated into a sustained release composition.

The compositions described herein can be prepared by known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable carrier. Suitable carriers are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985). On this basis, the compositions include, albeit not exclusively, solutions of the substances in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.

Recombinant nucleic acid molecules comprising a sense, an antisense sequence or oligonucleotide fragment thereof, may be directly introduced into cells or tissues in vivo using delivery vehicles known in the art such as retroviral vectors, adenoviral vectors and DNA virus vectors. They may also be introduced into cells in vivo using physical techniques known in the art such as microinjection and electroporation or chemical methods such as coprecipitation and incorporation of DNA into liposomes. Recombinant molecules may also be delivered in the form of an aerosol or by lavage.

The utility of the substances, antibodies, sense and antisense nucleic acid molecules, and compositions of the invention may be confirmed in animal experimental model systems. Suitable animal model systems which can be used to determine activity may include, but is not limited to retinoic acid or P450RAI-3 knock-out transgenic animals.

xv) Transgenic and Knock-Out Animals and Methods of Making Same

Nucleic acid molecules that encode P450RAI-3 or that encode proteins having a biological activity similar to that of a P450RAI-3, can be used to generate either transgenic animals or “knock-out” animals. These animals are useful in the development and screening of therapeutically useful reagents. A transgenic animal [e.g. a mouse] is an animal having cells that contain a transgene, which was introduced into the animal or an ancestor of the animal at prenatal, e.g. an embryonic stage. A transgene is a DNA molecule that has integrated into the genome of a cell from which a transgenic animal develops.

In one embodiment, a human P450RAI-3 cDNA, comprising the nucleotide sequence (SEQ. ID. NOS. 9 or 10), or an appropriate variant, fragment or sub-sequence thereof, can be used to generate transgenic animals that contain cells which express human P450RAI-3. Methods for generating transgenic animals, such as rats, hamsters, rabbits, sheep and pigs, and particularly mice, have become conventional in the art [see for example U.S. Pat. Nos. 4,736,866 and 4,870,009].

In a preferred embodiment, plasmids containing recombinant molecules of the present invention are microinjected into mouse embryos. In particular, the plasmids of the present invention are microinjected into the male pronuclei of fertilized one-cell mouse embryos, the injected embryos at the 2-4 cell stage are transferred to pseudo-pregnant foster females, and the embryos in the foster females are allowed to develop to term. [see Hogan et al., A Laboratory Manual, Cold Spring Harbour, N. Y. Cold Spring Harbour (1986)]

Alternatively, an embryonal stem cell line can be transfected with an expression vector comprising a polynucleotide encoding a protein having P450RAI-3 activity, and cells containing the polynucleotide can be used to form aggregation chimeras with embryos from a suitable recipient mouse stain. The chimeric embryos can be implanted into a suitable pseudopregnant female mouse of the appropriate strain and the embryo brought to term. Progeny harbouring the transfected DNA in their germ cells can be used to breed uniformly transgenic mice.

Transgenic animals that include a copy of a P450RAI-3 transgene introduced into the germ line of the animal by an embryonic stage can also be used to examine the effect of increased P450RAI-3 expression in various tissues.

Conversely, “knock-out” animals that have a defective or altered P450RAI-3 gene can be constructed [see for example Lemoine and Cooper, Gene Therapy, Human Molecular Genetics Series, BIOS Scientific Publishers, Oxford, U.K. (1996)]. Knock-out animals can be made that cannot express a functional P450RAI-3 polypeptide. For example, a portion of the of P450RAI DNA (e.g. an exon) can be deleted or replaced with another gene, such as a gene encoding a selectable marker, that can be used to monitor integration. The altered P450RAI-3 DNA can then be transfected into an embryonal stem cell line where it will homologously recombine with the endogenous P450RAI-3 gene in certain cells. Clones containing the altered gene can be selected. Cells containing the altered gene are injected into a blastocyst of an animal, such as a mouse, to form aggregation chimeras and chimeric embryos are implanted as described above for transgenic animals. Transmission of the altered gene into the germline of a resultant animal can be confirmed using standard techniques and the animal can be used to breed animals having an altered P450RAI-3 gene in every cell. Such a knock-out animal may be used, for example, to test the effectiveness of an agent in the absence of a P450RAI-3 protein, if lack of P450RAI-3 expression does not result in lethality. The knock-out animal can also be used to monitor the development of any conditions related to altered P450RAI-3 expression or activity.

The following non-limiting examples are illustrative of the present invention:

EXAMPLES Example 1 Determination of cDNA sequence encoding P450RAI-3

The Unfinished High Throughput Genomic Sequences (htgs) database at the National Center for Biotechnology Information (NCBI), available over the Internet at http://www.ncbi.nlm.nih.gov/BLAST/was searched using the amino acid sequences of human P450RAI-1 (SEQ. ID. NO. 2, or see FIG. 6A) and human P450RAI-2 (SEQ. ID. NO. 4 or see FIG. 6A). The TBLASTN algorithm of the Translated BLAST program was used to search the 6 possible reading frames of all the HTG sequences against the two protein query sequences. Parameters for all searches were the defaults of Blosum 62 which use a gap existence cost of 11, per residue cost of 1 and lambda ratio of 0.85. The nucleotide and the corresponding amino acid sequences, which showed similarity to SEQ. ID. NO. 2 or 4 (also see FIG. 6A) were retrieved from GenBank.

One of the subject sequences obtained from GenBank (AL358613), identified here as SEQ. ID. NO. 6, a 160,532 bp clone from human chromosome 10 (clone name RP11-348J12, library RPCl-11.2, sequencing ongoing at Sanger Genome Center; center project name: bA348J12, http://www.sanger.ac.uk) showed similarity to the nucleotide sequence encoding the amino acid sequences identified as SEQ. ID. NO. 2 and 4 (See FIG. 6A). As described below, the present inventor determined that this clone comprised within it the polynucleotide sequence encoding the novel cytochrome P450 of the invention, P450RAI-3.

Using the amino acid sequences of human P450RAI-1 (SEQ. ID. NO. 2, see also FIG. 6A) and human P450RAI-2 (SEQ. ID. NO. 4, See also FIG. 6A) aligned to the 6 possible reading frame translations of SEQ. ID. NO. 6 (in 43 unordered pieces), the N-terminal exon 1, as well as exons 4 and 5 and part of C-terminal exon 6 were predicted. Exon 6 contained the heme thiolate benchmark sequence characteristic of all P450s (FXXGXXXCXG, where X can be any amino acid, SEQ. ID. NO. 7), FGGGARSCLG (SEQ. ID. NO. 8).

A bacterial stab of the BAC clone (AL358613) was obtained from the Sanger Genome Center. Colonies were PCR screened using two sets of primers to identify positives containing the known fragments of P450RAI-3. One set was specific for the first predicted exon, 5′-CTCATCATGTTCCCTTGGGGGCTGA (SEQ. ID. NO. 25) (nucleotides 1-19 bp of SEQ. ID. NO. 10) and 5′-CTGCTGAACTMCCAGTGCAGCGTTTC (SEQ. ID. NO. 26) (complementary to nucleotides 204-181 bp of SEQ. ID. NO. 10). The other set was within predicted exon 4,5′-GCAAGGGACCAGCTGCATCGGCACCTG (SEQ. ID. NO. 27) (nucleotides 715-741 bp of SEQ. ID. NO. 10) and 5′-CCCTTGCACTGTGAATGATTAGGTCG (SEQ. ID. NO. 29) (complementary to nucleotides 826-801 bp of SEQ. ID. NO. 10). BAC clone DNA was prepared and sent to the Centre for Applied Genomics at the Hospital for Sick Children in Toronto, Ontario and the sequencing was performed to complete the genomic sequence around the identified regions containing P450RAI-3.

Using the contig sequence information from AL358613 and the sequence obtained from the Centre for Applied Genomics, a 22,179 bp piece of genomic DNA (SEQ. ID. NO. 9) was predicted. The nr database (all GenBank+EMBL+DDBJ+PDB sequences) was searched using SEQ. ID. NO. 9 using the BLASTN and BLASTX algorithms and a BLASTN and TBLASTX search was conducted against the htgs database. The nucleotide (SEQ. ID. NO. 10, also see FIG. 2) and resulting translated protein sequence (SEQ. ID. NO. 11, also see FIG. 3) of P450RAI-3 was identified. The intron/exon boundaries were deduced based on the loss of amino acid similarity between P450RAI-1, P450RAI-2 and the relevant regions of the genomic sequence. The 6 exons and the intron/exon boundaries of the novel P450, P450RAI-3 are shown in FIG. 5. The amino acids encoded within the respective exons are identified above the schematic diagram and nucleotide positions in relation to the human genomic sequence (SEQ. ID. NO. 9, also see FIG. 1) are provided below the diagram. However, it would be appreciated that the positions of the exons noted in FIG. 5 are approximate and may vary from the actual boundaries.

The sequence has been termed P450RAI-3 based on sequence homology with P450RAI-1 (CYP26A1) and P450RAI-2 (CYP26B1). The amino acid sequence comparison between human P450RAI-3 and human P450RAI-1 and P450RAI-2 is shown in FIG. 6A. Overall P450RAI-1 and P450RAI-3 show 43% identity at the amino acid level and 52% at the nucleotide level over the region of the predicted open reading frame. The overall similarity of the two putative open reading frames is somewhat higher when conservatively substituted amino acids are considered. Overall P450RAI-2 and P450RAI-3 show 51% identity at the amino acid level and 61% at the nucleotide level over the region of the predicted open reading frame. Again, the overall similarity of the two putative open reading frames is somewhat higher when conservatively substituted amino acids are considered.

EXAMPLE 2 P450RAI-3 Tissue Expression

In order to find tissues in which P450RAI-3 is expressed, a multi-tissue poly A+ RNA dot blot containing 76 different human tissues (Clontech, Calif.) was probed. A 200 bp PCR fragment (SEQ. ID. NO. 24) was amplified from peripheral blood leukocyte cDNA (Origene, Md.) using the primer set for the first predicted exon, SEQ. ID. NOS. 25 and 26. The random-primed a-[³²P]dATP-labeled probe was hybridized to the blot using conditions described by the manufacturer. FIG. 7A illustrates the results. A distinct signal of P450RAI-3 was detected in the adrenal gland. FIG. 7B depicts the tissue map of the 76 tissue samples.

Multi-tissue RACE cDNA panels (Origene, Md.) representing different human tissues (muscle, stomach, testis, placenta, pituitary gland, thyroid gland, adrenal gland and pancreas) were PCR-amplified using a set of primers specific for the first predicted exon of the putative P450RAI-3 gene (SEQ. ID. NOS. 25 and 26). Thus, any amplified product from P450RAI-3 should be the size of exon 1, or about 200 bp. PCR products were fractionated on 1% agarose gel and analyzed under UV. The results are shown in FIG. 8. From the figure it can be seen that the 200 bp amplified fragment is expressed again primarily in the adrenal gland, and to a lesser extent, was detected in testis.

P450RAI-3 expression in the adrenal gland was confirmed by the results illustrated in FIG. 9. Adrenal gland mRNA (Ambion, Tex.) was used for RT-PCR with a primer specific to the predicted exon 4 (SEQ. ID. NO. 27) and one within exon 6,5′-CTCGTGCGTGTCCCGGATGCTATAC (SEQ. ID. NO. 28) (complementary to nucleotides 1248-1224 bp of SEQ. ID. NO. 10) of P450RAI-3. Amplification was conducted in accordance to manufacturer's recommendations (Clontech, Calif.). Amplification products were fractionated on 1% agarose gel and visualized under ultraviolet light along side DNA markers (FIG. 9, panel A). The gel was then blotted on Hybond ECL nitrocellulose membrane (Amersham Pharmacia Biotech, UK) and fixed to the membrane by baking at 80° C. for 2 hours under vacuum. Prehybridization and hybridization steps were performed using ExpressHyb (Clontech, Calif.) according to the manufacturer's directions. The blot was probed with an end-labeled [g-[³²P]ATP using T4 polynucleotide kinase] internal exon 4-specific oligonucleotide, (SEQ. ID. NO. 29). The blot was washed two times for 15 minutes in 2×SSC, 0.1% SDS at room temperature then for 15 minutes at 60° C. in 0.1% SSC and 0.1% SDS and exposed at 70° C. overnight to Kodak X-OMAT AR film (Eastman Kodak Company, NY) (FIG. 9, panel B). A 0.5 kbp fragment corresponding to the predicted PCR amplified product from P450RAI-3 can be seen in FIG. 9, panel B.

EXAMPLE 3 All-Trans-Retinoic-Acid (ATRA)-Metabolism Activity:

Mammalian cell transfection: Enzymatic activity

Mammalian Cos-1 cells were transfected with either pcDNA-P450RAI-3 or pcDNA-control vector using Fugene™ (Roche diagnostic) according to the manufacturers directions. After 48 hours post-tranfection, cells were washed with PBS and assessed for ATRA-metabolism activity as described Briefly, transfected cells were incubated with radio-labeled ³[H]-ATRA (18000 cpm, 2 nM) for 3 hours. Organic and Water-soluble metabolites were extracted using Bligh-Dyer procedure and radioactivity counted (FIG. 10). HPLC analysis of the organic metabolites: the Bligh-Dyer organic button-layer was dried out and residues were suspended in HPLC solvent and then analyzed by HPLC as described in [White et al, PNAS, vol 97, pg 6403-6408], FIG. 11.

P450RAI-3 was found to efficiently metabolize ATRA to polar metabolites.

EXAMPLE 4 Substrate Competition in Cell Assay—Competition with Retinoid Isomers and Metabolites

The purpose of these experiments is to determine the specific substrates for P450RAI-3. Unlabeled ATRA, 9-cis, 13-cis-RA, 4-OH-RA, 4-Oxo-RA, 18-OH-RA Retinol, Retinal, and ketoconazole were used in [³H]-RA-metabolism inhibition assay as follows: P450-RAI stable transfected Hela cells were maintained in MEM medium containing 10% FBS and 100 μg/ml Hygromycin B. Cells were cultured in cell culture dishes, exponentially growing cells (70 to 80% confluents) were harvested, washed with PBS and then plated in a 48-well plate at 5×10⁵ cells per well in 0.150 ml MEM medium containing 10% FBS. Increasing concentration of either unlabeled ATRA, RA-precursors (Retinol, Retinal), RA-isomers (13-cis and 9-cis forms), or RA-metabolites (40H-RA, 4-OXORA, and 18-OHRA) were dissolved in DMSO and added to the cells. The RA metabolism reaction was started by the addition of 50 ul of [³H]-RA solution (20000 cpm, 2 nM final concentration). Cells were incubated for 3 hours in a humidified CO₂ incubator at 37 degrees Celsius. The reaction was stopped by the addition of 5 ul of 10% acetic acid. Organic and aqueous soluble radio-labelled metabolites were extracted using the Bligh-Dyer procedure.

Results were presented in FIG. 12.

ATRA was the best competing substrate (ID₅₀ 0.3 μM), then the RA-metabolites (40H-RA, and 4-OXO-RA). Surprisingly, even at lower concentrations 9-cis-RA was found to compete for P450RAI-3 (ID₅₀ 1 μM) and ketoconazole was a weak inhibitor (ID₅₀ 70 μM). However, as was determined for CYP26-A and CYP26-B from previous data, Retinol (Vitamin A) and Retinal were not substrates for CYP26—C.

Substrate competition: Comparison of P450RAI-3 vs. CYP26-B

Since 9-cis-RA was found to compete for P450RAI-3, we compared the [³H]-RA-metabolism inhibition was compared with increased concentrations of unlabeled ATRA, 9-cis-RA, 13-cis-RA and Ketoconazole in transient Cos-P450RAI-3 vs. Cos-CYP26-B-2 cell-based assay

Results are presented in FIG. 13 and table I

At lower concentrations, unlabeled ATRA competed for both P450RAI-3 and B, However 9-cis-RA isomer competed only for P450RAI-3. 13-cis-RA isomer was not a substrate for both. Ketoconazole inhibited CYP26-B but was only a weak inhibitor for P450RAI-3 (CYP26C). TABLE 1 P450RAI-3/B Transient Cos cell assay/ID₅₀ (_M) P450RAI-3 P450RAI-3 Substrate 1^(st) Assay 2^(nd) Assay CYP26-B ATRA 0.2 0.35 3.5 9cis-RA 1 1.2 >10 13cis-RA >10 >10 >10 Retinol >100 Retinal >100 Ketoconazole 70 50 7 4-OH-RA 0.2 4-OXO-RA 0.4 18-OH-RA 1

EXAMPLE 5 P450RAI-3 Expression in Insect Cells and Microsomes Preparation

Recombinant baculovirus expressing either P450RAI-3 or CYP26B (Bac-P450RAI-3 or Bac-CYP26B or CYP26B) was prepared and titrated by end-point dilution and infection of Sf9 cells in 96-well format. Baculovirus expressing vector was co-transfected into Sf9-insect cells. Infected cells were assessed for RA-metabolism after 72 hours post transfection using RA-metabolism cell based assay as follows: the RA-metabolism reaction was started by adding 50 μl of [³H]-RA solution (20000 cpm, 2 nM final concentration) using an automatic repeating-pipette.

Cells were incubated for 3 hours. The reaction was stopped by adding 5 μl of 10% acetic acid. Organic and aqueous soluble radio-labelled metabolites were extracted using the Bligh-Dyer procedure.

P450RAI-3 as well as CYP26-B-infected Sf9-cells metabolized ATRA and converted it to water-soluble metabolites, FIG. 14.

Microsomes from P450RAI-3 and CYP26B infected Sf-9 cells were made as follows: exponentially growing cells were harvested, washed with ice-cold PBS, counted then homogenized and sonicated in lysis buffer containing protease inhibitors cocktail (10⁸ cells par 5 ml lyses buffer). Since P450RAI-3 is a microsomal cytochrome, it preferably uses NADPH cytochromic P450 reductase and a flavoprotein for activity. The microsomes were isolated by differential centrifugations (10 min at 800×g, than 10 min at 10,000×g and finally the postmitichondirial supernatant was centrifuged at 100,000×g for 60 min using ultracentrifuge (Beckman). The microsomal pellet was isolated and homogenized gently in the storage buffer containing protease inhibitors cocktail. Protein concentration in the microsomal preparation was determined using a Bradford reaction assay kit with BSA as standards (Pierce). The absorbance at 562 nm of standards (BSA at 1, 0.5, 0.25, 0.125, and 0.0625 mg/ml) and 3 dilutions of microsomes samples (1/5, 1/10 and 1/20) was determined using an automatic microplate reader (μQuant, BioTek instruments Inc.). The protein concentration directly calculated by the software (KCJunior). The microsomes were aliquoted at 1 mg/ml in storage buffer, checked for enzymatic activity, and than stored in liquid nitrogen until further use. All procedures carried out at 4° C. Microsomes were assessed for P450 content by carbon monoxide assay and total P450 content was determined at 3 nmol/ml.

RA-metabolism activity was assessed using RA-metabolism microsomes-based assay and Bligh Dyer extraction as follows: Using an automatic repeating-pipette, gently thawed and homogenized microsomes were plated in a 48-well plate in final volume of 150 μl of storage buffer containing 0.5 mg/ml BSA. At indicated concentration 2 μl of compound solution was added to the assay and 2 μl of DMSO was added to MS control (quadruplet) and No-MS control. 50 μl of [³H]-RA solution (30,000 cpm, 2 nM final concentration) was added. The microsomes assay with the compounds and the [³H]-ATRA were incubated first for 10 min at 37° C., and then the reaction started by adding 20 μl of NADPH solution (or 1 mM final). After 1-hour incubation the reaction was stopped by adding 5 μl of 10% acetic acid. Organic and aqueous soluble radio-labelled metabolites were extracted using the Bligh-Dyer procedure. Saturated activity obtained in presence of NADPH at 5 μg of microsomes, FIG. 14.

EXAMPLE 6 Substrate Competition in Microsomes Assay: Comparison of P450RAI-3 vs. CYP26-B

Sf9-Insect microsomes assay using ATRA, 9-cis-RA and 13-cis-RA competition assay using Sf9-Bac-P450RAI-3 insect microsomes: P450RAI-3 vs. CYP26-B.

Since 9-cis-RA competed for P450-RAI-3-RA metabolism the [³H]-RA-metabolism inhibition with increase concentration of either unlabeled ATRA, 9-cis-RA, 13-cis-RA or Ketoconazole in Sf9-Bac-P450RAI-3 and in Sf9-Bac-CYP26-B insect microsomes was compared.

Results presented in FIG. 15 and ID₅₀ values in table 2:

Unlabeled ATRA competed for both P450RAI-3 and CYP26-B with ID₅₀ values of 20 and 30 nM respectively. However, 9-cis-RA competed only for P450RAI-3 (ID₅₀ 80 nM), a very weak 9-cis-RA competition can be observed for CYP26-B at much higher concentrations isomer are a high affinity substrates for P450RAI-3. TABLE 2 Summary of ID₅₀ values ID₅₀ (nM) Insect Sf9- ID₅₀ (nM) microsomes P450RAI-3 Sf9-CYP26-B ATRA   20  32 9cisRA   86 5000 13cis-RA  3000 4500 Ketoconazole >10000  750 Substrate competition in Mammalian microsomes: P450RAI-3 vs. CYP26-B

Microsomes prepared from either stable Hela-CYP26-B or transient P450RAI-53-transfected Cos cells were used. [³H]-RA-metabolism inhibition assay was carried out with increased concentration of unlabeled ATRA, 13-ci-sRA and 9-cis-RA isomers as described in example 5.

Results are presented in table 3 and FIG. 16.

Mammalian microsomes gave similar results previously obtained with microsomes prepared from Baculovirus Sf9 infected insect cells. Briefly, ATRA was the highest affinity substrate for CYP26-B and P450RAI-3 (ID₅₀ 20 nM). However 9cis-RA competed for P450RAI-3 (ID₅₀ 80 nM) but not CYP26B. As expected, 13-cis-RA did not compete at lower concentrations for either CYP26A or CYP 26B. Ketoconazole a weak inhibitor of P450RAI-3 (ID₅₀ 70 μM, instead of ID₅₀ 0.7 μM for CYP26B). TABLE 3 Summary of ID₅₀ values in mammalian microsomes ID50 (nM) Mammalian P450RAI-3 CYP26-B ATRA   20  20 9cisRA   85 4000 13cis-RA  4000 2000 Ketoconazole 72000  740

Example 7 ATRA and 9-cis-RA Binding Assay

Binding experiments of ATRA to P450RAI-3 were done twice-using Sf9-Bac-P450RAI-3 insect microsomes. Result presented in FIG. 17, and ID₅₀ values in table 4.

Experiment was carried out along with CYP26-B and Kd values of 48 nM and 114 nM were obtained for P450RAI-3 and CYP26-B, respectively. The second time P450RAI-3 was assayed alone and Kd value of 42 nM was obtained. Competition of ATRA binding to P450RAI-3 by ketoconazole was also done twice. The first time ketoconazole concentration of up to 10 μM was used but only 15% inhibition was obtained at the highest concentration. The second time the assay was done along with CYP26-B and ketoconazole concentration of up to 100 μM was used. Kd values of 1.5 μM and 70.5 μM were obtained for P450RAI-2 and P450RAI-3, respectively.

Binding of 9-cis-RA to CYP26-A, B, and C was also carried out at substrate concentrations of 0.05 nM to 1000 nM, FIG. 18.

Very good binding was only observed with P450RAI-3 microsomes. However, weak binding of 9-cis-RA to CYP26-A and CYP26-B was also observed. While Kd value of 69 nM was obtained for P450RAI-3, it was not possible to determine the Kd values for CYP26-A and CYP26-B because binding of 9-cis-RA to both proteins was linear for substrate concentration up to 1000 nM. Kd values were summarized in table 4

As was found with the enzymatic competition assay, the binding assay confirmed the high affinity of ATRA as well as 9-cis-RA for P450RAI-3 TABLE 4 Summary of Kd values P450RAI-3 CYP2-6B CYP26-A ATRA 45 100 ND 9cisRA 69 ND ND Ketoconazole 75 1.5 ND

Example 8 P450RAI-3 Metabolism of 9-cis-RA

Since 9-cis-RA was found to compete and bind to P450RAI-3, we conducted studies of 9-cis-RA-metabolism in P450RAI-3 transient transfected Cos cells. Mammalian Cos-1 cells were transfected with either pcDNA-P450RAI-3 or pcDNA-control vector according to the manufacturers directions using Fugene (Roche). After 48 hours post-tranfection, cells were washed with PBS and assessed for 9-cis-RA-metabolism activity as described using Bligh-Dyer procedure. Briefly, transfected cells were incubated with radio-labeled ³[H]-9-cis-RA (38000 cpm, 2 nM) for 3 hours. Organic and Water-soluble metabolites were extracted using Bligh-Dyer procedure and radioactivity counted, FIG. 19.

HPLC analysis of 9-cis-RA metabolism: the button Organic layer was dried out and residues were suspended in HPLC solvent and then analyzed by HPLC as described [White et al, PNAS, vol 97, pg. 6403], FIG. 20.

Results: P450RAI-3-tranfected Cos cells converted 9-cis-RA to the hydoxy and oxo-metabolites and to more polar water-soluble metabolites.

Example 9 P450RAI-3: LC/MS Analysis of ATRA and 9-cis-RA Metabolism Pathway

Hela-P450RAI-3 stable cells were used to determine the ATRA and 9-cis-RA metabolism pathway. Hela-P450RAI-3 cells were incubated with either 1 _M ATRA or 9-cis-RA in 10 ml MEM media containing 10% FBS. After 5 hours incubation at 37° C., cells were acidified with acetic acid and total retinoids were extracted using Ethyl acetate method. The organic upper layer was pooled and evaporated in speed-vacuum, and dried residues were dissolved in HPLC mobile solvent and analyzed in LC/MS.

The HPLC system consisted of a waters alliance 2690 separations module. Chromatography was obtained on an Eclipse X DB-C18 reverse phase column (5 um, 2.1×150 mm from HP). A gradient elution program was used using acetonitrile, water and 10% acetic acid. The flow was held at 0.2 ml/min. The UV absorbance was set at 351 and used to monitor retinoid standards (ATRA, 9-cis-RA, 13-cis-RA, 4-OH-RA, 18-OH-RA, 4-Oxo-RA-RA). Mass Spectrometry condition used for this work consisted of a Micro-mass Quatro Ultima Tandem triple quadruple mass spectrometer equipped with electrospray interface (ESI) and operated in a negative mode. Two method for MS mode MS and MS/MS, 1) A Full scan mode from m/z 200-500 was used and 2) And MS/MS mode using a precursors ion scan mode was used to screen and identify the Hydroxy, and Oxo-metabolite peaks of RA, Results presented in FIG. 21

P450RAI-3 metabolized ATRA as well as 9-cis-RA to hydroxy and oxo-metabolites.

Example 10 Stable Mammalian Cell Expressing P450RAI-3

Cell Transfection and Cloning

Hela cells were transfected with either pcDNA or pcEBV-vector containing P450RAI-3 and hygromycin resistance gene. Then the transfected cells were selected in MEM media containing 10% FBS and Hygromycin 200_g/ml. Surviving selected cells were tested for RA-activity and cloned. Two high activity clones (pcEBVclone #1, pcDNA-clone#22) were selected, expanded assessed for ATRA and 9-cis-RA metabolism (FIG. 22).

Microsomes were made from Hela-P450RAI-3 clone 1 and assessed for RA-metabolism activity and then stored in liquid nitrogen (FIG. 23).

Mammalian cells which stably express CYP26C have been generated and shown to possess ATRA and 9-cis RA metabolic activity.

While the present invention has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the invention is not limited to the disclosed examples. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

REFERENCES

Particulars of references cited above are given below. All of the listed references are incorporated herein by refer11ence.

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1-72. (Cancelled)
 73. An isolated nucleic acid molecule comprising a polynucleotide comprising a sequence selected from the group consisting of: (a) SEQ. ID. NO. 10 or encoding the amino acid sequence of SEQ. ID. NO. 11; (b) a polynucleotide of (a) wherein T can also be U: (c) a polynucleotide having a nucleic acid sequence which differs from any of the nucleic acid molecules of (a) to (c) in codon due to the degeneracy of the genetic code; and (d) a polynucleotide that is a variant, allelic variant or encodes a species homologue of any one of the polynucleotides of (a) to (c).
 74. An isolated nucleic acid molecule comprising a fragment of the nucleic acid molecule of claim 73 and which will hybridize thereto under stringent hybridization conditions wherein the polynucleotide is at least 15 bases and does not hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence of only A residues or of only T residues.
 75. An isolated nucleic acid molecule of claim 74, wherein the polynucleotide fragment comprises a nucleotide sequence selected from SEQ. ID NOS. 12, 14, 16, 18, 20 and 22 or encoding the amino acid sequence selected from SEQ. ID. NOS. 13, 15, 17, 19, 21 and 23 or variant thereof.
 76. An isolated nucleic acid molecule having a nucleic acid sequence complementary to the nucleic acid molecule of claim
 73. 77. An isolated nucleic acid molecule of claim 73 encoding a cytochrome P450 retinoid metabolizing protein, comprising SEQ. ID. NO. 10 or a nucleic acid molecule encoding the amino acid sequence of SEQ. ID. NO. 11 or a variant thereof or biologically active fragment thereof.
 78. The isolated nucleic acid molecule of claim 77, wherein the retinoid is all trans retinoic acid(ATRA) or 9-cis-retinoic acid.
 79. An isolated nucleic acid molecule of that is 90% homologous to that of claim
 77. 80. The isolated nucleic acid molecule of claim 73, wherein the nucleotide sequence comprises sequential nucleotide deletions from either the C-terminus or the N-terminus.
 81. A recombinant vector comprising an isolated nucleic acid molecule of claim
 73. 82. A recombinant host cell comprising an isolated nucleic acid molecule of claim
 73. 83. The recombinant host cell of claim 82 wherein the isolated nucleic acid molecule is operatively linked to a regulatory sequence to allow expression of a peptide encoded by said nucleic acid sequence.
 84. An isolated polypeptide comprising an amino acid sequence at least 95% homologous to a sequence selected from the group consisting of: (a) a polypeptide comprising the amino acid sequence of SEQ. ID. NO. 11; (b) a polypeptide encoded by any of the polynucleotides selected from the group consisting of: i. a polynucleotide encoding a polypeptide comprising amino acid sequence of SEQ. ID. NO. 13, 15, 17, 19, or 21; ii. a polynucleotide consisting of the nucleotide sequence of SEQ. ID. NO. 10: iii. a polynucleotide of SEQ. ID. NO. 10 wherein T can also be U; iv. a polynucleotide that is a variant, allelic variant or encodes a species homologue of any one of the polynucleotides of (i) to (iii). (c) a mature form, variant, allelic variant or species homologue to any of the polypeptides of (a) or (b); (d) fragment of any of the polypeptides of (a) to (c).
 85. The isolated polypeptide of claim 84 that is a cytochrome P450 retinoid metabolizing polypeptide comprising amino acid sequence, SEQ. ID. NO. 11, a variant thereof or a biologically active fragment thereof.
 86. The isolated polypeptide of claim 85, wherein the retinoid is all-trans retinoic acid or 9-cis retinoic acid.
 87. The isolated polypeptide of claim 85 consisting of the amino acid sequence of SEQ. ID. NO. 11 (FIG. 3).
 88. An isolated antibody that binds specifically to the isolated polypeptide of claim 84 or immunogenic or antigenic portion thereof.
 89. A microsome comprising the isolated polypeptide of claim
 84. 90. A method of making an isolated polypeptide of claim 84 comprising: (e) culturing a recombinant host cell comprising a nucleic acid molecule encoding said polypeptide operatively linked to a regulatory sequence to enable expression of said polypeptide, under conditions such that said polypeptide is expressed; and (f) recovering said polypeptide.
 91. A pharmaceutical composition comprising the isolated polypeptide of claim 85, and/or optionally a modulator of P450RAI-3 activity in combination with a pharmaceutically acceptable carrier.
 92. The pharmaceutical composition of claim 91, further comprising an adjuvant.
 93. A method for preventing, treating or ameliorating a medical condition related to P450RAI-3 expression which comprises administering to a mammalian subject a therapeutically effective amount of the polypeptide of claim 85 or a polynucleotide encoding for said polypeptide and/or a modulator of P450RAI-3.
 94. The method of claim 93, wherein the modulator of P450RAI-3 is an inhibitor and the inhibitor is administered with a P450RAI-3 substrate.
 95. The method of claim 94, wherein the substrate is all trans retinoic acid and/or 9-cis retinoic acid.
 96. A method of diagnosis of a P450RAI-3-related condition or a predisposition to a P450RAI-3-related condition comprising detecting a polymorphism in a P450RAI-3 gene, wherein detection of said polymorphism is indicative of the occurrence of said condition or a predisposition thereto.
 97. The method of claim 96 wherein the condition is related to vitamin A or retinoic acid metabolism.
 98. A diagnostic Kit for identification of polymorphisms in the P450RAI-3 gene, comprising a nucleic acid molecule of claim 73 or a nucleic acid molecule complimentary thereto, and optionally directions for the method comprising screening the P450RAI-3 gene from a human for polymorphisms, wherein detection of said polymorphisms is indicative of the occurrence of a P450RAI-3-related condition or a predisposition thereto.
 99. A method of treating a disease or condition related to vitamin A or retinoic acid metabolism in a patient comprising administering to the patient in need thereof, an effective amount of a therapeutically effective polypeptide of claim 85 and/or an agonist or antagonist thereof.
 100. A method of identifying modulators of P450RAI-3 activity in a biological assay, wherein the method comprises: [a]expressing P450RAI-3 in a cell or obtaining P450RAI-3 in a microsome or in vitro: [b] adding a substrate under conditions that enable P450RAI-3 activity; and [c] detecting activity of P450RAI-3 on said substrate in the presence or absence of a modulator.
 101. The method of claim 100, wherein the substrate is ATRA and/or 9-cis RA.
 102. The method of claim 101, wherein the substrate comprises radio-labeled ATRA or 9-cis-RA.
 103. A method of determining the ATRA and/or 9-cis-RA metabolizing activity of a polypeptide of claim 84, comprising: expressing the polypeptide in a host cell, adding ATRA and/or 9-cis-RA to the cell, and determining the amount anchor rate of ATRA and/or 9-cis-RA metabolism.
 104. The method of claim 103, wherein the ATRA comprises radio-labeled ATRA and the 9-cis-RA comprises radio-labeled 9-cis-RA.
 105. The method of claim 103, wherein the amount and/or rate of ATRA and 9-cis-RA metabolism is determined by measuring the amount and/or rate of production of hydoxy-metabolites and oxo-metabolites.
 106. The method of claim 103, wherein prior to the step adding ATRA and/or 9-cis-RA to the cell, a candidate activator or inhibitor of the polypeptide is added to the cell.
 107. A method of identifying the substrate of a polypeptide of claim 85, comprising: expressing the polypeptide in a host cell, adding a candidate substrate, and determining if the substrate is metabolized, wherein metabolization indicates that the candidate substrate is a substrate of the polypeptide.
 108. The method of claim 107, wherein a plurality of candidate substrates are added to the cell to determine the amount and rate of metabolism of each substrate.
 109. The method of claim 108, wherein, prior to the step adding the candidate substrates to the cell, a candidate activator or inhibitor of the polypeptide is added to the cell.
 110. A method of determining the binding activity of a substrate to a polypeptide of claims 85, comprising expressing the polypeptide in a host cell, adding a candidate substrate, and determining a Kd value.
 111. The method of claim 110, wherein prior to the step adding the substrate to the cell, a candidate activator or inhibitor of the polypeptide is added to the cell.
 112. A method for conducting a drug discovery and/or a pharmaceutical business; (k) providing one or more assay systems for identifying agents by their ability to modulate P450RAI-3 activity or expression or retinoic acid metabolism; (l) conducting therapeutic profiling of agents identified in step (a), or further analogs thereof, for efficacy and toxicity in animals; and (m)formulating a pharmaceutical preparation including one or more agents identified in step (b) as having an acceptable therapeutic profile.
 113. The method of claim 112, including a step of establishing a distribution system for distributing the pharmaceutical preparation for sale.
 114. The method of claim 113, including establishing a sales group for marketing the pharmaceutical preparation.
 115. A method of conducting a target discovery business comprising. i. providing one or more assay systems for identifying agents by their ability to modulate P450RAI-3 and/or retinoic acid metabolism; ii. (optionally) conducting therapeutic profiling of agents identified in step (a) for efficacy and toxicity in animals; and iii. licensing, to a third party, the rights for further drug development and/or sales for agents identified in step (a), or analogs thereof. 